Peptides and t cells for use in immunotherapeutic treatment of various cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/320,763, filed May 14, 2021, which is a continuation of U.S. patentapplication Ser. No. 17/017,358, filed Sep. 10, 2020, now U.S. Pat. No.11,065,316, issued Jul. 20, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/887,765, filed May 29, 2020, now U.S. Pat. No.10,898,557 issued Jan. 26, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/673,619, filed Nov. 4, 2019, now U.S. Pat. No.10,695,411, issued Jun. 30, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/982,293, filed May 17, 2018, now U.S. Pat. No.10,576,132, issued Mar. 3, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/249,083, filed Aug. 26, 2016, now U.S. Pat. No.10,335,471, issued Jul. 2, 2019, which claims the benefit of U.S.Provisional Application Ser. No. 62/211,276, filed Aug. 28, 2015, andGreat Britain Application No. 1515321.6, filed Aug. 28, 2015, thecontent of each of these applications is herein incorporated byreference in their entirety.

This application also is related to PCT/EP2016/070146 filed 26 Aug.2016, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2912919-054015_Sequence_listing_ST25.txt” createdon Feb. 7, 2022, and 65,367 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO), cancer ranged amongthe four major non-communicable deadly diseases worldwide in 2012. Forthe same year, colorectal cancer, breast cancer and respiratory tractcancers were listed within the top 10 causes of death in high incomecountries.

Epidemiology

In 2012, 14.1 million new cancer cases, 32.6 million patients sufferingfrom cancer (within 5 years of diagnosis) and 8.2 million cancer deathswere estimated worldwide (Ferlay et al., 2013; Bray et al., 2013).

Within the groups of brain cancer, leukemia and lung cancer, the presentapplication particularly focuses on glioblastoma (GB), chroniclymphocytic leukemia (CLL), and non-small cell and small cell lungcancer (NSCLC and SCLC).

Lung cancer is the most common type of cancer worldwide and the leadingcause of death from cancer in many countries.

Breast cancer is an immunogenic cancer entity and different types ofinfiltrating immune cells in primary tumors exhibit distinct prognosticand predictive significance. A large number of early phase immunotherapytrials have been conducted in breast cancer patients. Most of thecompleted vaccination studies targeted HER2 and carbohydrate antigenslike MUC-1 and revealed rather disappointing results. Clinical data onthe effects of immune checkpoint modulation with ipilimumab and other Tcell-activating antibodies in breast cancer patients are emerging(Emens, 2012).

Chronic Lymphocytic Leukemia

While CLL is not curable at present, many patients show only slowprogression of the disease or worsening of symptoms. As patients do notbenefit from an early onset of treatment, the initial approach is “watchand wait” (Richards et al., 1999). For patients with symptomatic orrapidly progressing disease, several treatment options are available.These include chemotherapy, targeted therapy, immune-based therapieslike monoclonal antibodies, chimeric antigen-receptors (CARs) and activeimmunotherapy, and stem cell transplants.

Monoclonal antibodies are widely used in hematologic malignancies. Thisis due to the knowledge of suitable antigens based on the goodcharacterization of immune cell surface molecules and the accessibilityof tumor cells in blood or bone marrow. Common monoclonal antibodiesused in CLL therapy target either CD20 or CD52. Rituximab, the firstmonoclonal anti-CD20 antibody originally approved by the FDA fortreatment of NHLs, is now widely used in CLL therapy. Combinationaltreatment with rituximab/fludarabine/cyclophosphamide leads to higher CRrates and improved overall survival (OS) compared to the combinationfludarabine/cyclophosphamide and has become the preferred treatmentoption (Hallek et al., 2008). Ofatumomab targets CD20 and is used fortherapy of refractory CLL patients (Wierda et al., 2011). Obinutuzumabis another monoclonal anti-CD20 antibody used in first-line treatment incombination with chlorambucil (Goede et al., 2014).

Alemtuzumab is an anti-CD52 antibody used for treatment of patients withchemotherapy-resistant disease or patients with poor prognostic factorsas del 17p or p53 mutations (Parikh et al., 2011).

Novel monoclonal antibodies target CD37 (otlertuzumab, BI 836826,IMGN529 and (177)Lu-tetulomab) or CD40 (dacetuzumab and lucatumumab) andare tested in pre-clinical settings (Robak and Robak, 2014).

Several completed and ongoing trials are based on engineered autologouschimeric antigen receptor (CAR)-modified T cells with CD19 specificity(Maus et al., 2014). So far, only the minority of patients showeddetectable or persistent CARs. One partial response (PR) and twocomplete responses (CR) have been detected in the CAR T-cell trials byPorter et al. and Kalos et al. (Kalos et al., 2011; Porter et al.,2011).

Active immunotherapy includes the following strategies: gene therapy,whole modified tumor cell vaccines, DC-based vaccines and tumorassociated antigen (TAA)-derived peptide vaccines.

Approaches in gene therapy make use of autologous genetically modifiedtumor cells. These B-CLL cells are transfected withimmuno-(co-)stimulatory genes like IL-2, IL-12, TNF-alpha, GM-CSF, CD80,CD40L, LFA-3 and ICAM-1 to improve antigen presentation and T cellactivation (Carballido et al., 2012). While specific T-cell responsesand reduction in tumor cells are readily observed, immune responses areonly transient.

Several studies have used autologous DCs as antigen presenting cells toelicit anti-tumor responses. DCs have been loaded ex vivo with tumorassociated peptides, whole tumor cell lysate and tumor-derived RNA orDNA. Another strategy uses whole tumor cells for fusion with DCs andgeneration of DC—B-CLL-cell hybrids. Transfected DCs initiated both CD4+and CD8+ T-cell responses (Muller et al., 2004). Fusion hybrids and DCsloaded with tumor cell Iysate or apoptotic bodies increasedtumor-specific CD8+ T-cell responses. Patients that showed a clinicalresponse had increased IL-12 serum levels and reduced numbers of Tregs(Palma et al., 2008).

Different approaches use altered tumor cells to initiate or increaseCLL-specific immune responses. An example for this strategy is thegeneration of trioma cells: B-CLL cells are fused to anti-Fc receptorexpressing hybridoma cells that have anti-APC specificity. Trioma cellsinduced CLL-specific T-cell responses in vitro (Kronenberger et al.,2008).

Another strategy makes use of irradiated autologous CLL cells withBacillus Calmette-Guérin as an adjuvant as a vaccine. Several patientsshowed a reduction in leukocyte levels or stable disease (Hus et al.,2008).

Besides isolated CLL cells, whole blood from CLL patients has been usedas a vaccine after preparation in a blood treatment unit. The vaccineelicited CLL-specific T-cell responses and led to partial clinicalresponses or stable disease in several patients (Spaner et al., 2005).

Several TAAs are over-expressed in CLL and are suitable forvaccinations. These include fibromodulin (Mayr et al., 2005),RHAMM/CD168 (Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006), hTERT(Counter et al., 1995), the oncofetal antigen-immature laminin receptorprotein(OFAiLRP) (Siegel et al., 2003), adipophilin (Schmidt et al.,2004), survivin (Granziero et al., 2001), KW1 to KW14 (Krackhardt etal., 2002) and the tumor-derived IgVHCDR3 region (Harig et al., 2001;Carballido et al., 2012). A phase I clinical trial was conducted usingthe RHAMM-derived R3 peptide as a vaccine. 5 of 6 patients haddetectable R3-specific CD8+ T-cell responses (Giannopoulos et al.,2010).

Colorectal Cancer

Depending on the colorectal cancer (CRC) stage, different standardtherapies are available for colon and rectal cancer. Standard proceduresinclude surgery, radiation therapy, chemotherapy and targeted therapyfor CRC (Berman et al., 2015a; Berman et al., 2015b).

In addition to chemotherapeutic drugs, several monoclonal antibodiestargeting the epidermal growth factor receptor (EGFR, cetuximab,panitumumab) or the vascular endothelial growth factor-A (VEGF-A,bevacizumab) are administered to patients with high stage disease. Forsecond-line and later treatment the inhibitor for VEGF aflibercept, thetyrosine kinase inhibitor regorafenib and the thymidylate-synthetaseinhibitor TAS-102 and the dUTPase inhibitor TAS-114 can be used(Stintzing, 2014; Wilson et al., 2014).

The most recent clinical trials analyze active immunotherapy as atreatment option against CRC. Those strategies include the vaccinationwith peptides from tumor-associated antigens (TAAs), whole tumor cells,dendritic cell (DC) vaccines and viral vectors (Koido et al., 2013).

Peptide vaccines have so far been directed against carcinoembryonicantigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognizedby T cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG),Wilms' Tumor antigen 1 (WT1), Survivin-2B, MAGE3, p53, ring fingerprotein 43 and translocase of the outer mitochondrial membrane 34(TOMM34), or mutated KRAS. In several phase I and II clinical trialspatients showed antigen-specific CTL responses or antibody production.In contrast to immunological responses, many patients did not benefitfrom peptide vaccines on the clinical level (Koido et al., 2013; Miyagiet al., 2001; Moulton et al., 2002; Okuno et al., 2011).

Dendritic cell vaccines comprise DCs pulsed with either TAA-derivedpeptides, tumor cell lysates, apoptotic tumor cells, or tumor RNA orDC-tumor cell fusion products. While many patients in phase I/II trialsshowed specific immunological responses, only the minority had aclinical benefit (Koido et al., 2013).

Whole tumor cell vaccines consist of autologous tumor cells modified tosecrete GM-CSF, modified by irradiation or virus-infected, irradiatedcells. Most patients showed no clinical benefit in several phase II/IIItrials (Koido et al., 2013).

Vaccinia virus or replication-defective avian poxvirus encoding CEA aswell as B7.1, ICAM-1 and LFA-3 have been used as vehicles in viralvector vaccines in phase I clinical trials. A different study usednonreplicating canarypox virus encoding CEA and B7.1. Besides theinduction of CEA-specific T cell responses 40% of patients showedobjective clinical responses (Horig et al., 2000; Kaufman et al., 2008).

Esophageal Cancer

The primary treatment strategy for esophageal cancer depends on tumorstage and location, histological type and the medical condition of thepatient. Surgery alone is not sufficient, except in a small subgroup ofpatients with squamous cell carcinoma.

Data on immunotherapeutic approaches in esophageal cancer are scarce, asonly a very limited number of early phase clinical trials have beenperformed. A vaccine consisting of three peptides derived from threedifferent cancer-testis antigens (TTK protein kinase, lymphocyte antigen6 complex locus K and insulin-like growth factor (IGF)-II mRNA bindingprotein 3) was administered to patients with advanced esophageal cancerin a phase I trial with moderate results. Intra-tumoral injection ofactivated T cells after in vitro challenge with autologous malignantcells elicited complete or partial tumor responses in four of elevenpatients in a phase I/II study (Toomey et al., 2013).

Gastric Cancer

Gastric cancer (GC) begins in the cells lining the mucosal layer andspreads through the outer layers as it grows. Surgery is the primarytreatment and the only curative treatment for gastric cancer. Theefficacy of current therapeutic regimens for advanced GC is poor,resulting in low 5-year survival rates. Immunotherapy might be analternative approach to ameliorate the survival of GC patients. Adoptivetransfer of tumor-associated lymphocytes and cytokine induced killercells, peptide-based vaccines targeting HER2/neu, MAGE-3 or vascularendothelial growth factor receptor 1 and 2 and dendritic cell-basedvaccines targeting HER2/neu showed promising results in clinical GCtrials. Immune checkpoint inhibition and engineered T cells mightrepresent additional therapeutic options, which is currently evaluatedin pre-clinical and clinical studies (Matsueda and Graham, 2014).

Glioblastoma

The therapeutic options for glioblastoma (WHO grade IV) are verylimited. According to the guidelines released by the German Society forNeurology the standard therapy in young patients includes resection orbiopsy of the tumor, focal radiation therapy and chemotherapy withtemozolomide or CCNU/lomustine or a combination of procarbazine withCCNU and vincristine (PCV). In the USA, Canada and Switzerland treatmentwith bevacizumab (anti-VEGF-antibody) is also approved for relapsetherapy (Leitlinien fiar Diagnostik und Therapie in der Neurologie,2014).

Different immunotherapeutic approaches are investigated for thetreatment of GB, including immune-checkpoint inhibition, vaccination andadoptive transfer of engineered T cells.

Antibodies directed against inhibitory T cell receptors or their ligandswere shown to efficiently enhance T cell-mediated anti-tumor immuneresponses in different cancer types, including melanoma and bladdercancer. The effects of T cell activating antibodies like ipilimumab andnivolumab are therefore assessed in clinical GB trials, but preliminarydata indicate autoimmune-related adverse events.

Different vaccination strategies for GB patients are currentlyinvestigated, including peptide-based vaccines, heat-shock proteinvaccines, autologous tumor cell vaccines, dendritic cell-based vaccinesand viral protein-based vaccines. In these approaches peptides derivedfrom GB-associated proteins like epidermal growth factor receptorvariant III (EGFRvIII) or heat shock proteins or dendritic cells pulsedwith autologous tumor cell lysate or cytomegalo virus components areapplied to induce an anti-tumor immune response in GB patients. Severalof these studies reveal good safety and tolerability profiles as well aspromising efficacy data.

Adoptive transfer of genetically modified T cells is an additionalimmunotherapeutic approach for the treatment of GB. Different clinicaltrials currently evaluate the safety and efficacy of chimeric antigenreceptor bearing T cells directed against HER2, IL-13 receptor alpha 2and EGFRvIII (Ampie et al., 2015).

Liver Cancer

Disease management depends on the tumor stage at the time of diagnosisand the overall condition of the liver. If surgery is not a treatmentoption, different other therapies are available at hand.

Lately, a limited number of immunotherapy trials for HCC have beenconducted. Cytokines have been used to activate subsets of immune cellsand/or increase the tumor immunogenicity (Reinisch et al., 2002; Sangroet al., 2004). Other trials have focused on the infusion ofTumor-infiltrating lymphocytes or activated peripheral blood lymphocytes(Shi et al., 2004; Takayama et al., 1991; Takayama et al., 2000b).

So far, a small number of therapeutic vaccination trials have beenexecuted. Butterfield et al. conducted two trials using peptides derivedfrom alpha-fetoprotein (AFP) as a vaccine or DCs loaded with AFPpeptides ex vivo (Butterfield et al., 2003; Butterfield et al., 2006).In two different studies, autologous dendritic cells (DCs) were pulsedex vivo with autologous tumor lysate (Lee et al., 2005) or lysate of thehepatoblastoma cell line HepG2 (Palmer et al., 2009). So far,vaccination trials have only shown limited improvements in clinicaloutcomes.

Melanoma

The standard therapy in melanoma is complete surgical resection withsurrounding healthy tissue. If resection is not complete or not possibleat all, patients receive primary radiation therapy, which can becombined with interferon-alpha administration in advanced stages (stagesIIB/C and IIIA-C).

Enhancing the anti-tumor immune responses appears to be a promisingstrategy for the treatment of advanced melanoma. In the United Statesthe immune checkpoint inhibitor ipilimumab as well as the BRAF kinaseinhibitors vemurafenib and dabrafenib and the MEK inhibitor trametinibare already approved for the treatment of advanced melanoma. Bothapproaches increase the patient's anti-tumor immunity—ipilimumabdirectly by reducing T cell inhibition and the kinase inhibitorsindirectly by enhancing the expression of melanocyte differentiationantigens. Additional checkpoint inhibitors (nivolumab and lambrolizumab)are currently investigated in clinical studies with first encouragingresults. Additionally, different combination therapies targeting theanti-tumor immune response are tested in clinical trials includingipilimumab plus nivolumab, ipilimumab plus a gp100-derived peptidevaccine, ipilimumab plus dacarbazine, ipilimumab plus IL-2 and iplimumabplus GM-CSF (Srivastava and McDermott, 2014).

Several different vaccination approaches have already been evaluated inpatients with advanced melanoma. So far, phase III trials revealedrather disappointing results and vaccination strategies clearly need tobe improved. Therefore, new clinical trials, like the OncoVEX GM-CSFtrial or the DERMA trial, aim at improving clinical efficacy withoutreducing tolerability.

Adoptive T cell transfer shows great promise for the treatment ofadvanced stage melanoma.

In vitro expanded autologous tumor infiltrating lymphocytes as well as Tcells harboring a high affinity T cell receptor for the cancer-testisantigen NY-ESO-1 had significant beneficial and low toxic effects upontransfer into melanoma patients. Unfortunately, T cells with highaffinity T cell receptors for the melanocyte specific antigens MART1 andgp100 and the cancer-testis antigen MAGEA3 induced considerable toxiceffects in clinical trials. Thus, adoptive T cell transfer has hightherapeutic potential, but safety and tolerability of these treatmentsneeds to be further increased (Phan and Rosenberg, 2013; Hinrichs andRestifo, 2013).

Non-Small Cell Lung Cancer

Treatment options are determined by the type (small cell or non-smallcell) and stage of cancer and include surgery, radiation therapy,chemotherapy, and targeted biological therapies such as bevacizumab,erlotinib and gefitinib.

To expand the therapeutic options for NSCLC, different immunotherapeuticapproaches have been studied or are still under investigation. Whilevaccination with L-BLP25 or MAGEA3 failed to demonstrate anvaccine-mediated survival advantage in NSCLC patients, an allogeneiccell line-derived vaccine showed promising results in clinical studies.Additionally, further vaccination trials targeting gangliosides, theepidermal growth factor receptor and several other antigens arecurrently ongoing. An alternative strategy to enhance the patient'santi-tumor T cell response consists of blocking inhibitory T cellreceptors or their ligands with specific antibodies. The therapeuticpotential of several of these antibodies, including ipilimumab,nivolumab, pembrolizumab, MPDL3280A and MEDI-4736, in NSCLC is currentlyevaluated in clinical trials (Reinmuth et al., 2015).

Ovarian Cancer

Surgical resection is the primary therapy in early as well as advancedstage ovarian carcinoma. Surgical removal is followed by systemicchemotherapy with platinum analogs, except for very low grade ovariancancers (stage IA, grade 1), where post-operative chemotherapy is notindicated.

Immunotherapy appears to be a promising strategy to ameliorate thetreatment of ovarian cancer patients, as the presence ofpro-inflammatory tumor infiltrating lymphocytes, especially CD8-positiveT cells, correlates with good prognosis and T cells specific fortumor-associated antigens can be isolated from cancer tissue.

Therefore, a lot of scientific effort is put into the investigation ofdifferent immunotherapies in ovarian cancer. A considerable number ofpre-clinical and clinical studies has already been performed and furtherstudies are currently ongoing. Clinical data are available for cytokinetherapy, vaccination, monoclonal antibody treatment, adoptive celltransfer and immunomodulation.

Cytokine therapy with interleukin-2, interferon-alpha, interferon-gammaor granulocyte-macrophage colony stimulating factor aims at boosting thepatient's own anti-tumor immune response and these treatments havealready shown promising results in small study cohorts.

Phase I and II vaccination studies, using single or multiple peptides,derived from several tumor-associated proteins (Her2/neu, NY-ESO-1, p53,Wilms tumor-1) or whole tumor antigens, derived from autologous tumorcells revealed good safety and tolerability profiles, but only low tomoderate clinical effects.

Monoclonal antibodies that specifically recognize tumor-associatedproteins are thought to enhance immune cell-mediated killing of tumorcells. The anti-CA-125 antibodies oregovomab and abagovomab as well asthe anti-EpCAM antibody catumaxomab achieved promising results in phaseII and III studies. In contrast, the anti-MUC1 antibody HMFG1 failed toclearly enhance survival in a phase III study.

An alternative approach uses monoclonal antibodies to target and blockgrowth factor and survival receptors on tumor cells. Whileadministration of trastuzumab (anti-HER2/neu antibody) and MOv18 andMORAb-003 (anti-folate receptor alpha antibodies) only conferred limitedclinical benefit to ovarian cancer patients, addition of bevacizumab(anti-VEGF antibody) to the standard chemotherapy in advanced ovariancancer appears to be advantageous.

Adoptive transfer of immune cells achieved heterogeneous results inclinical trials. Adoptive transfer of autologous, in vitro expandedtumor infiltrating T cells was shown to be a promising approach in apilot trial. In contrast, transfer of T cells harboring a chimericantigen receptor specific for folate receptor alpha did not induce asignificant clinical response in a phase I trial. Dendritic cells pulsedwith tumor cell lysate or tumor-associated proteins in vitro were shownto enhance the anti-tumor T cell response upon transfer, but the extentof T cell activation did not correlate with clinical effects. Transferof natural killer cells caused significant toxicities in a phase IIstudy.

Intrinsic anti-tumor immunity as well as immunotherapy are hampered byan immunosuppressive tumor microenvironment. To overcome this obstacleimmunomodulatory drugs, like cyclophosphamide, anti-CD25 antibodies andpegylated liposomal doxorubicin are tested in combination withimmunotherapy. Most reliable data are currently available foripilimumab, an anti-CTLA4 antibody, which enhances T cell activity.Ipilimumab was shown to exert significant anti-tumor effects in ovariancancer patients (Mantia-Smaldone et al., 2012).

Pancreatic Cancer

Therapeutic options for pancreatic cancer patients are very limited. Onemajor problem for effective treatment is the typically advanced tumorstage at diagnosis. Additionally, pancreatic cancer is rather resistantto chemotherapeutics, which might be caused by the dense andhypovascular desmoplastic tumor stroma.

According to the guidelines released by the German Cancer Society, theGerman Cancer Aid and the Association of the Scientific MedicalSocieties in Germany, resection of the tumor is the only availablecurative treatment option.

Vaccination strategies are investigated as further innovative andpromising alternative for the treatment of pancreatic cancer.Peptide-based vaccines targeting KRAS mutations, reactive telomerase,gastrin, survivin, CEA and MUC1 have already been evaluated in clinicaltrials, partially with promising results. Furthermore, clinical trialsfor dendritic cell-based vaccines, allogeneic GM-CSF-secreting vaccinesand algenpantucel-L in pancreatic cancer patients also revealedbeneficial effects of immunotherapy. Additional clinical trials furtherinvestigating the efficiency of different vaccination protocols arecurrently ongoing (Salman et al., 2013).

Prostate Cancer

The therapeutic strategy for prostate cancer mainly depends on thecancer stage. The dendritic cell-based vaccine sipuleucel-T was thefirst anti-cancer vaccine to be approved by the FDA. Due to its positiveeffect on survival in patients with CRPC, much effort is put into thedevelopment of further immunotherapies. Regarding vaccinationstrategies, the peptide vaccine prostate-specific antigen (PSA)-TRICOM,the personalized peptide vaccine PPV, the DNA vaccine pTVG-HP and thewhole cell vaccine expressing GM-CSF GVAX showed promising results indifferent clinical trials. Furthermore, dendritic cell-based vaccinesother than sipuleucel-T, namely BPX-101 and DCVAC/Pa were shown toelicited clinical responses in prostate cancer patients. Immunecheckpoint inhibitors like ipilimumab and nivolumab are currentlyevaluated in clinical studies as monotherapy as well as in combinationwith other treatments, including androgen deprivation therapy, localradiation therapy, PSA-TRICOM and GVAX. The immunomodulatory substancetasquinimod, which significantly slowed progression and increasedprogression free survival in a phase II trial, is currently furtherinvestigated in a phase III trial. Lenalidomide, anotherimmunomodulator, induced promising effects in early phase clinicalstudies, but failed to improve survival in a phase III trial. Despitethese disappointing results further lenalidomide trials are ongoing(Quinn et al., 2015).

Renal Cell Carcinoma

Initial treatment is most commonly either partial or complete removal ofthe affected kidney(s) and remains the mainstay of curative treatment(Rini et al., 2008). The known immunogenity of RCC has represented thebasis supporting the use of immunotherapy and cancer vaccines inadvanced RCC. The interesting correlation between lymphocytes PD-1expression and RCC advanced stage, grade and prognosis, as well as theselective PD-L1 expression by RCC tumor cells and its potentialassociation with worse clinical outcomes, have led to the development ofnew anti PD-1/PD-L1 agents, alone or in combination with anti-angiogenicdrugs or other immunotherapeutic approaches, for the treatment of RCC(Massari et al., 2015). In advanced RCC, a phase III cancer vaccinetrial called TRIST study evaluates whether TroVax (a vaccine using atumor-associated antigen, 5T4, with a pox virus vector), added tofirst-line standard of care therapy, prolongs survival of patients withlocally advanced or mRCC. Median survival had not been reached in eithergroup with 399 patients (54%) remaining on study however analysis of thedata confirms prior clinical results, demonstrating that TroVax is bothimmunologically active and that there is a correlation between thestrength of the 5T4-specific antibody response and improved survival.Further there are several studies searching for peptide vaccines usingepitopes being over-expressed in RCC.

Various approaches of tumor vaccines have been under investigation.Studies using whole-tumor approaches, including tumor cell lysates,fusions of dendritic cells with tumor cells, or whole-tumor RNA weredone in RCC patients, and remissions of tumor lesions were reported insome of these trials (Avigan et al., 2004; HoltI et al., 2002; Marten etal., 2002; Su et al., 2003; Wittig et al., 2001).

Small Cell Lung Cancer

The treatment and prognosis of SCLC depend strongly on the diagnosedcancer stage. Immune therapy presents an excessively investigated fieldof cancer therapy. Various approaches are studded in the treatment ofSCLC. One of the approaches targets the blocking of CTLA-4, a naturalhuman immune suppressor. The inhibition of CTLA-4 intends to boost theimmune system to combat the cancer. Recently, the development ofpromising immune check point inhibitors for treatment of SCLC has beenstarted. Another approach is based on anti-cancer vaccines which iscurrently available for treatment of SCLC in clinical studies (AmericanCancer Society, 2015; National Cancer Institute, 2015).

Acute Myeloid Leukemia

One treatment option for AML is targeting CD33 with antibody-drugconjugates (anti-CD33+calechiamicin, SGN-CD33a, anti-CD33+actinium-225),bispecific antibodies (recognition of CD33+CD3 (AMG 330) or CD33+CD16)and chimeric antigen receptors (CARs) (Estey, 2014).

Non-Hodgkin Lymphoma

Treatment of NHL depends on the histologic type and stage (NationalCancer Institute, 2015): Spontaneous tumor regression can be observed inlymphoma patients. Therefore, active immunotherapy is a therapy option(Palomba, 2012).

An important vaccination option includes Id vaccines. B lymphocytesexpress surface immunoglobulins with a specific amino acid sequence inthe variable regions of their heavy and light chains, unique to eachcell clone (=idiotype, Id). The idiotype functions as a tumor associatedantigen.

Passive immunization includes the injection of recombinant murineanti-Id monoclonal antibodies alone or in combination with IFNalpha, IL2or chlorambucil.

Active immunization includes the injection of recombinant protein (Id)conjugated to an adjuvant (KLH), given together with GM-CSF as an immuneadjuvant. Tumor-specific Id is produced by hybridoma cultures or usingrecombinant DNA technology (plasmids) by bacterial, insect or mammaliancell culture.

Three phase III clinical trials have been conducted (Biovest, Genitope,Favrille). In two trials patients had received rituximab. GM-CSF wasadministered in all three trials. Biovest used hybridoma-producedprotein, Genitope and Favrille used recombinant protein. In all threetrials Id was conjugated to KLH. Only Biovest had a significant result.

Vaccines other than Id include the cancer-testis antigens MAGE, NY-ESO1and PASD-1, the B-cell antigen CD20 or cellular vaccines. The latestmentioned consist of DCs pulsed with apoptotic tumor cells, tumor celllysate, DC-tumor cell fusion or DCs pulsed with tumor-derived RNA.

In situ vaccination involves the vaccination with intra-tumoral CpG incombination with chemotherapy or irradiated tumor cells grown in thepresence of GM-CSF and collection/expansion/re-infusion of T cells.

Vaccination with antibodies that alter immunologic checkpoints arecomprised of anti-CD40, anti-OX40, anti-41BB, anti-CD27, anti-GITR(agonist antibodies that directly enhance anti-tumor response) oranti-PD1, anti-CTLA-4 (blocking antibodies that inhibit the checkpointthat would hinder the immune response). Examples are ipilimumab(anti-CTLA-4) and CT-011 (anti-PD1) (Palomba, 2012).

Uterine cancer Treatment of endometrial carcinomas is stage-dependent.The majority of endometrical carcinomas comprises of well to moderatelydifferentiated endometrioid adenocarcinomas which are usually confinedto the corpus uteri at diagnosis and can be cured by hysterectomy (WorldCancer Report, 2014).

Also therapies for cervical cancer depend on the stage. In early stages,excision is the standard therapy which might be combined withradio-(chemo-)therapy. Primary radio-(chemo-)therapy is chosen at latestages (Stage III and higher), in cases with lymph node infiltration orin cases in which the tumor can not be excised.

There are also some immunotherapeutic approaches that are currentlybeing tested. In a Phase I/II Clinical Trial patients suffering fromuterine cancer were vaccinated with autologous dendritic cells (DCs)electroporated with Wilms' tumor gene 1 (WT1) mRNA. Besides one case oflocal allergic reaction to the adjuvant, no adverse side effects wereobserved and 3 out of 6 patients showed an immunological response(Coosemans et al., 2013).

As stated above, HPV infections provoke lesions that may ultimately leadto cervical cancer. Therefore, the HPV viral oncoproteins E6 and E7 thatare are constitutively expressed in high-grade lesions and cancer andand are required for the onset and maintenance of the malignantphenotype are considered promising targets for immunotherapeuticapproaches (Hung et al., 2008; Vici et al., 2014). One study performedAdoptive T-cell therapy (ACT) in patients with metastatic cervicalcancer. Patients receive an infusion with E6 and E7 reactivetumor-infiltrating T cells (TILs) resulting in complete regression in 2and a partial response in 1 out of 9 patients (Stevanovic et al., 2015).Furthermore, an intracellular antibody targeting E7 was reported toblock tumor groth in mice (Accardi et al., 2014). Also peptide, DNA andDC-based vaccines targing HPV E6 and E7 are in clinical trials (Vici etal., 2014).

Gallbladder Adenocarcinoma and Cholangiocarcinoma

Cholangiocarcinoma is mostly identified in advanced stages because it isdifficult to diagnose. Cholangiocarcinoma is difficult to treat and isusually lethal.

Gallbladder cancer is the most common and aggressive malignancy of thebiliary tract worldwide.

Urinary Bladder Cancer

The standard treatment for bladder cancer includes surgery, radiationtherapy, chemotherapy and immunotherapy.

At stage 0 and I, the bladder cancer is typically treated bytransurethral resection potentially followed by intravesicalchemotherapy and optionally combined with intravesical immunotherapeutictreatment with BCG (bacillus Calmette-Guérin).

An effective immunotherapeutic approach is established in the treatmentof aggressive non-muscle invasive bladder cancer (NMIBC). Thereby, aweakened form of the bacterium Mycobacterium bovis (bacillusCalmette-Guérin=BCG) is applied as an intravesical solution. The majoreffect of BCG treatment is a significant long-term (up to 10 years)protection from cancer recurrence and reduced progression rate. Inprinciple, the treatment with BCG induces a local inflammatory responsewhich stimulates the cellular immune response. The immune response toBCG is based on the following key steps: infection of urothelial andbladder cancer cells by BCG, followed by increased expression ofantigen-presenting molecules, induction of immune response mediated viacytokine release, induction of antitumor activity via involvement ofvarious immune cells (thereunder cytotoxic T lymphocytes, neutrophils,natural killer cells, and macrophages) (Fuge et al., 2015; Gandhi etal., 2013).

BCG treatment is in general well tolerated by patients but can be fatalespecially by the immunocompromised patients. BCG refractory is observedin about 30-40% of patients (Fuge et al., 2015; Steinberg et al.,2016a). The treatment of patients who failed the BCG therapy ischallenging. The patients who failed the BCG treatment are at high riskfor developing of muscle-invasive disease. Radical cystectomy is thepreferable treatment option for non-responders (Steinberg et al., 2016b;von Rundstedt and Lerner, 2015). The FDA approved second line therapy ofBCG-failed NMIBC for patients who desire the bladder preservation is thechemotherapeutic treatment with valrubicin. A number of other secondline therapies are available or being currently under investigation aswell, thereunder immunotherapeutic approaches like combinedBCG-interferon or BCG-check point inhibitor treatments, pre-BCGtransdermal vaccination, treatment with Mycobacterium phlei cellwall-nucleic acid (MCNA) complex, mono- or combination chemotherapy withvarious agents like mitomycin C, gemcitabine, docetaxel, nab-paclitaxel,epirubicin, mitomycin/gemcitabine, gemcitabine/docetaxel, anddevice-assisted chemotherapies like thermochemo-, radiochemo-,electromotive or photodynamic therapies (Fuge et al., 2015; Steinberg etal., 2016b; von Rundstedt and Lerner, 2015). Further evaluation ofavailable therapies in clinical trials is still required.

The alternative treatment options for advanced bladder cancer are beinginvestigated in ongoing clinical trials. The current clinical trialsfocused on the development of molecularly targeted therapies andimmunotherapies. The targeted therapies investigate the effects ofcancerogenesis related pathway inhibitors (i.e. mTOR, vascularendothelial, fibroblast, or epidermal growth factor receptors,anti-angiogenesis or cell cycle inhibitors) in the treatment of bladdercancer. The development of molecularly targeted therapies remainschallenging due to high degree of genetic diversity of bladder cancer.The main focus of the current immunotherapy is the development ofcheckpoint blockage agents like anti-PD1 monoclonal antibody andadoptive T-cell transfer (Knollman et al., 2015b; Grivas et al., 2015;Jones et al., 2016; Rouanne et al., 2016).

Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinomas (HNSCC) are heterogeneous tumorswith differences in epidemiology, etiology and treatment (Economopoulouet al., 2016).

HNSCC is considered an immunosuppressive disease, characterized by thedysregulation of immunocompetent cells and impaired cytokine secretion(Economopoulou et al., 2016). Immunotherapeutic strategies differbetween HPV-negative and HPV-positive tumors.

In HPV-positive tumors, the viral oncoproteins E6 and E7 represent goodtargets, as they are continuously expressed by tumor cells and areessential to maintain the transformation status of HPV-positive cancercells. Several vaccination therapies are currently under investigationin HPV-positive HNSCC, including DNA vaccines, peptide vaccines andvaccines involving dendritic cells (DCs). Additionally, an ongoing phaseII clinical trial investigates the efficacy of lymphodepletion followedby autologous infusion of TILs in patients with HPV-positive tumors(Economopoulou et al., 2016).

In HPV-negative tumors, several immunotherapeutic strategies arecurrently used and under investigation. The chimeric IgG1 anti-EGFRmonoclonal antibody cetuximab has been approved by the FDA incombination with chemotherapy as standard first line treatment forrecurring/metastatic HNSCC. Other anti-EGFR monoclonal antibodies,including panitumumab, nimotuzumab and zalutumumab, are evaluated inHNSCC. Several immune checkpoint inhibitors are investigated in clinicaltrials for their use in HNSCC. They include the following antibodies:Ipilimumab (anti-CTLA-4), tremelimumab (anti-CTLA-4), pembrolizumab(anti-PD-1), nivolumab (anti-PD-1), durvalumab (anti-PD-1), anti-KIR,urelumab (anti-CD137), and anti-LAG-3.

Two clinical studies with HNSCC patients evaluated the use of DCs loadedwith p53 peptides or apoptotic tumor cells. The immunological responseswere satisfactory and side effects were acceptable.

Several studies have been conducted using adoptive T cell therapy (ACT).T cells were induced against either irradiated autologous tumor cells orEBV. Results in disease control and overall survival were promising(Economopoulou et al., 2016).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and hepatocellular carcinoma (HCC),colorectal carcinoma (CRC), glioblastoma (GB), gastric cancer (GC),esophageal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer(PC), renal cell carcinoma (RCC), benign prostate hyperplasia (BPH),prostate cancer (PCA), ovarian cancer (OC), melanoma, breast cancer,chronic lymphocytic leukemia (CLL), Merkel cell carcinoma (MCC), smallcell lung cancer (SCLC), Non-Hodgkin lymphoma (NHL), acute myeloidleukemia (AML), gallbladder cancer and cholangiocarcinoma (GBC, CCC),urinary bladder cancer (UBC), uterine cancer (UEC), head and necksquamous cell carcinoma (HNSCC), in particular. There is also a need toidentify factors representing biomarkers for cancer in general and theabove-mentioned cancer types in particular, leading to better diagnosisof cancer, assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.

This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated peptides of the invention can act as MHC class II activeepitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated und thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 388 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 388, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

While the most important criterion for a peptide to function as cancertherapy target is its over-presentation on primary tumor tissues ascompared to normal tissues, also the RNA expression profile of thecorresponding gene can help to select appropriate peptides.Particularly, some peptides are hard to detect by mass spectrometry,either due to their chemical properties or to their low copy numbers oncells, and a screening approach focusing on detection of peptidepresentation may fail to identify these targets. However, these targetsmay be detected by an alternative approach starting with analysis ofgene expression in normal tissues and secondarily assessing peptidepresentation and gene expression in tumors. This approach was realizedin this invention using an mRNA database (Lonsdale, 2013) in combinationwith further gene expression data (including tumor samples), as well aspeptide presentation data. If the mRNA of a gene is nearly absent innormal tissues, especially in vital organ systems, targeting thecorresponding peptides by even very potent strategies (such asbispecific affinity-optimized antibodies or T-cell receptors), is morelikely to be safe. Such peptides, even if identified on only a smallpercentage of tumor tissues, represent interesting targets. Routine massspectrometry analysis is not sensitive enough to assess target coverageon the peptide level. Rather, tumor mRNA expression can be used toassess coverage. For detection of the peptide itself, a targeted massspectrometry approach with higher sensitivity than in the routinescreening may be necessary and may lead to a better estimation ofcoverage on the level of peptide presentation.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 388 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 388,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1A andTable 2A bind to HLA-A*02. Peptides in Table 1C and Table 2B bind toHLA-A*24. Peptides in Table 1B bind to HLA class II alleles. Thepeptides in Table 3 are additional peptides that are HLA-A*24 bindingand may be useful in combination with the other peptides of theinvention.

TABLE 1A Peptides according to the present invention, HLA-A*02-binding.SEQ ID No. Sequence GeneID(s) Official Gene Symbol(s)  1 PLWGKVFYL    10926 DBF4  2 ALYGKLLKL    157680 VPS13B  3 TLLGKQVTL    157680VPS13B  4 ELAEIVFKV 203427, 349075, 51373 SLC25A43, ZNF713, MRPS17  5SLFGQEVYC     10840 ALDH1L1  6 FLDPAQRDL     57677 ZFP14  7 AAAAKVPEV    23382 AHCYL2  8 KLGPFLLNA 100508781, 653199, 9747 FAM115B, FAM115A 9 FLGDYVENL     54832 VPS13C 10 KTLDVFNIIL     54832 VPS13C 11GVLKVFLENV 121504, 554313, HIST4H4, HIST2H4B, 12 GLIYEETRGV8294, 8359, 8360, HIST1F4I, HIST1H4A, 13 VLRDNIQGI 8361, 8362, 8363,HIST1H4D, HIST1H4F, 8364, 8365, 8366, HIST1H4K, HIST1H4J,8367, 8368, 8370 HIST1H4C, HIST1H4H, HIST1H4B, HIST1H4E,HIST1H4L, HIST2H4A 14 LLDHLSFINKI     64863 METTL4 15 ALGDYVHAC     4588 MUC6 16 HLYNNEEQV 101060798, 1645, 8644 AKR1C1, AKR1C3      17 ILHEHHIFL      4233 MET 18 YVLNEEDLQKV      4233 MET 19 TLLPTVLTL   127707 KLHDC7A 20 ALDGHLYAI    127707 KLHDC7A 21 SLYHRVLLY     57221KIAA1244 22 MLSDLTLQL     57221 KIAA1244 23 AQTVVVIKA101059911, 4586, 727897 MUC5AC, MUC5B 24 FLWNGEDSAL 4586, 727897MUC5AC, MUC5B 25 IQADDFRTL 101059911, 4586, 727897 MUC5AC, MUC5B 26KVDGVVIQL 101059911, 4586, 727897 MUC5AC, MUC5B 27 KVFGDLDQV    169611OLFML2A 28 TLYSMDLMKV    169611 OLFML2A 29 TLCNKTFTA     26137 ZBTB20 30TVIDECTRI     26137 ZBTB20 31 ALSDETKNNWEV      5591 PRKDC 32 ILADEAFFSV     5591 PRKDC 33 LLLPLLPPLSPSLG    347252 IGFBPL1 34 LLPKKTESHHKT8330, 8331 HIST1H2AK, HIST1H2AJ 35 YVLPKLYVKL 100128168, 100996747,RPS26P39, RPS26P11, 441502, 6231, 643003, RPS26, RPS26P28,644166, 644928, 644934, RPS26P20, RSP26915, 646753, 728937, 729188RPS26P50, RPS26P2, RPS26P25, RPS26P58 36 KLYGIEIEV     56107 PCDHGA9 37ALINDILGELVKL     85463 ZC3H12C 38 KMQEDLVTL       781 CACNA2D1 39ALMAVVSGL     55103 RALGPS2 40 SLLALPQDLQA 1364, 1365, 23562,CLDN4, CLDN3, CLDN14, 9074, 9080 CLDN6, CLDN9 41 FVLPLVVTL      2848GPR25 42 VLSPFILTL    113730 KLHDC7B 43 LLWAGPVTA     28603 TRBV6-4 44GLLWQIIKV      5357 PLS1 45 VLGPTPELV 100124692 46 SLAKHGIVAL     10693CCT6B 47 GLYQAQVNL     89886 SLAMF9 48 TLDHKPVTV    203447 NRK 49LLDESKLTL     64097 EPB41L4A 50 EYALLYHTL        26 ABP1 51 LLLDGDFTL   347051 SLC10A5 52 ELLSSIFFL    160418 TMTC3 53 SLLSHVIVA       545ATR 54 FINPKGNWLL      3673 ITGA2 55 IASAIVNEL     57448 BIRC6 56KILDLTRVL     79783 C7orf10 57 VLISSTVRL    166379 BBS12 58 ALDDSLTSL     2302 FOXJ1 59 ALTKILAEL    339766 MROH2A 60 FLIDTSASM 203522, 26512DDX26B, INTS6 61 HLPDFVKQL      9857 CEP350 62 SLFNQEVQI100528032, 22914, 8302 KLRK1, KLRC4 63 TLSSERDFAL 100293534, 720, 721C4A, C4B 64 GLSSSSYEL     89866 SEC16B 65 KLDGICWQV       733 C8G 66FITDFYTTV     80055 PGAP1 67 GVIETVTSL     79895 ATP8B4 68 ALYGFFFKI   118663 BTBD16 69 GIYDGILHSI 158809, 392433 MAGEB6, MAGEB6P1 70GLFSQHFNL      1789 DNMT3B 71 GLITVDIAL     84162 KIAA1109 72 GMIGFQVLL6006, 6007 RHCE, RHD 73 GVPDTIATL     23120 ATP10B 74 ILDETLENV   167227 DCP2 75 ILDNVKNLL      4602 MYB 76 ILLDESNFNHFL    222584FAM83B 77 IVLSTIASV 10559, 154313 SLC35A1, C6orf165 78 LLWGHPRVA    25878 MXRA5 79 SLVPLQILL 101060288, 101060295, PRAMEF5, PRAMEF9,101060308, 343068, 343070, PRAMEF4, PRAMEF11, 400735, 440560, 440561,PRAMEF6, PRAMEF15, 441873, 645359, PRAMEF23 6533619, 729368 80 TLDEYLTYL101060308, 343068, PRAMEF5, PRAMEF9, 343070, 653619 PRAMEF15 81VLFLGKLLV    204962 SLC44A5 82 VLLRVLIL       102 ADAM10 83 ELLEYLPQL     5288 PIK3C2G 84 FLEEEITRV      6570 SLC18A1 85 STLDGSLHAV      2081ERN1 86 LLVTSLVVV 118471, 118472 PRAP1, ZNF511 87 YLTEVFLHVV     55024BANK1 88 ILLNTEDLASL    388015 RTL1 89 YLVAHNLLL      9365 KL 90GAVAEEVLSSI    340273 ABCB5 91 SSLEPQIQPV     23029 RBM34 92 LLRGPPVARA     3486 IGFBP3 93 SLLTQPIFL    151295 SLC23A3

TABLE 1BPeptides according to the present invention, HLA class 11 binding.SEQ ID No. Sequence GeneID(s) Official Gene Symbol(s) 94LKMENKEVLPQLVDAVTS  4547 MTTP 95 GLYLPLFKPSVSTSKAIGGGP 10165 SLC25A13

TABLE 1C Peptides according to the present invention, HLA-A*24 binding.SEQ ID No. Sequence GeneID(s) Official Gene Symbol(s)  96 YYTQYSQTI 25878 MXRA5  97 TYTFLKETF 203238 CCDC171  98 VFPRLHNVLF   9816 URB2  99QYILAVPVL  91147 TMEM67 100 VYIESRIGTSTSF  10112 KIF20A 101 IYIPVLPPHL163486 DENND1B 102 VYPFENFEF 127700 OSCP1 103 NYIPVKNGKQF   3096 HIVEP1104 SYLTWHQQI 125919 ZNF543 105 IYNETITDLL   1062 CENPE 106 IYNETVRDLL  3833 KIFC1 107 KYFPYLVVI  80131 LRRC8E 108 PYLVVIHTL  80131 LRRC8E 109LFITGGQFF 114134 SLC2A13 110 SYPKIIEEF   2177 FANCD2 111 VYVQILQKL  4998 ORC1 112 IYNFVESKL   4998 ORC1 113 IYSFHTLSF  55183 RIF1 114QYLDGTWSL  55083 KIF26B 115 RYLNKSFVL  63926 ANKRD5 116 AYVIAVHLF  10178TENM1 117 IYLSDLTYI  55103 RALGPS2 118 KYLNSVQYI  55103 RALGPS2 119VYRVYVTTF  57089 ENTPD7 120 GYIEHFSLW   5069 PAPPA 121 RYGLPAAWSTF 79713 IGFLR1 122 EYQARIPEF  55758 RCOR3 123 VYTPVLEHL   5591 PRKDC 124TYKDYVDLF   5591 PRKDC 125 VFSRDFGLLVF   5591 PRKDC 126 PYDPALGSPSRLF389058 SP5 127 QYFTGNPLF   3237 HOXD11 128 VYPFDWQYI   7941 PLA2G7 129KYIDYLMTW 55233, 92597 MOB1A, MOB1B 130 VYAHIYHQHF 55233, 92597MOB1A, MOB1B 131 EYLDRIGQLFF  51608 GET4 132 RYPALFPVL  11237 RNF24 133KYLEDMKTYF   5273 SERPINB10 134 AYIPTPIYF  81796 SLCO5A1 135 VYEAMVPLF 85465 EPT1 136 IYPEWPVVFF  51146 A4GNT 137 EYLHNCSYF 25909, 285116AHCTF1, AHCTF1P1 138 VYNAVSTSF  79915 ATAD5 139 IFGIFPNQF  79895 ATP8B4140 RYLINSYDF  84002 B3GNT5 141 SYNGHLTIWF  56245 C21orf62 142 VYVDDIYVI 57082 CASC5 143 KYIFQLNEI 347475 CCDC160 144 VFASLPGFLF   1233 CCR4 145VYALKVRTI   1237 CCR8 146 NYYERIHAL   8832 CD84 147 LYLAFPLAF 253782CERS6 148 SYGTVSQIF  23601 CLEC5A 149 SYGTVSQI  23601 CLEC5A 150IYITRQFVQF  81501 DCSTAMP 151 AYISGLDVF   8632 DNAH17 152 KFFDDLGDELLF  8632 DNAH17 153 VYVPFGGKSMITF 146754 DNAH2 154 VYGVPTPHF 151651 EFHB155 IYKWITDNF   2302 FOXJ1 156 YYMELTKLLL  51659 GINS2 157 DYIPASGFALF 84059 GPR98 158 IYEETRGVLKVF 121504, 554313, HIST4H4, HIST2H4B,8294, 8359, 8360, HIST1H4I, HIST1H4A, 8361, 8362, 8363,HIST1H4D, HIST1H4F, 8364, 8365, 8366, HIST1H4K, HIST1H4J,8367, 8368, 8370 HIST1H4C, HIST1H4H, HIST1H4B, HIST1H4E,HIST1H4L, HIST2H4A 159 IYEETRGVL 8367, 8368, 8370 HIST1H4L, HIST2H4A 160RYGDGGSSF   3188 HNRNPH2 161 KYPDIVQQF  29851 ICOS 162 KYTSYILAF   3458IFNG 163 RYLTISNLQF  28785 IGLV4-60 164 HYVPATKVF 259307 IL4I1 165EYFTPLLSGQF  55175 KLHL11 166 FYTLPFHLI  55175 KLHL11 167 RYGFYYVEF197021 LCTL 168 RYLEAALRL  10609 LEPREL4 169 NYITGKGDVF  84125 LRRIQ1170 QYPFHVPLL   4049 LTA 171 NYEDHFPLL   4109 MAGEA10 172 VFIFKGNEF  4319 MMP10 173 QYLEKYYNL   4319 MMP10 174 VYEKNGYIYF   4322 MMP13 175LYSPVPFTL 387521 TMEM189 176 FYINGQYQF  55728 N4BP2 177 VYFKAGLDVF254827 NAALADL2 178 NYSSAVQKF   4983 OPHN1 179 TYIPVGLGRLL  58495 OVOL2180 KYLQVVGMF   5021 OXTR 181 VYPPYLNYL   5241 PGR 182 AYAQLGYLLF   9033PKD2L1 183 PYLQDVPRI  92340 C17orf72 184 IYSVGAFENF 389677 RBM12B 185QYLVHVNDL  23322 RPGRIP1L 186 VFTTSSNIF  10371 SEMA3A 187 AYAANVHYL151473 SLC16A14 188 GYKTFFNEF  64078 SLC28A3 189 AYFKQSSVF  54790 TET2190 LYSELTETL  54790 TET2 191 TYPDGTYTGRIF 201633 TIGIT 192 RYSTFSEIF  8277 TKTL1 193 LYLENNAQTQF   8626 TP63 194 VYQSLSNSL 286827 TRIM59 195AYIKGGWIL 125488 TTC39C 196 GYIRGSWQF  79465 ULBP3 197 IFTDIFHYL  54464XRN1 198 DYVGFTLKI     19 ABCA1 199 SYLNHLNNL 154664 ABCA13 200VFIHHLPQF 116285 ACSM1 201 GYNPNRVFF 158067 AK8 202 RYVEGIVSL    246ALOX15 203 VYNVEVKNAEF  84250 ANKRD32 204 EYLSTCSKL 196528 ARID2 205VYPVVLNQ I  79798 ARMC5 206 NYLDVATFL  10973 ASCC3 207 LYSDAFKFIVF344905 ATP13A5 208 TYLEKIDGF 100526740, 26024, 9551ATP5J2-PTCD1, PTCD1, ATP5J2 209 AFIETPIPLF    631 BFSP1 210 IYAGVGEFSF   701 BUB1B 211 VFKSEGAYF 375444 C5orf34 212 SYAPPSEDLF100533105, 23678 SGK3 213 SYAPPSEDLFL 100533105, 23678 SGK3 214KYLMELTLI   9133 CCNB2 215 SYVASFFLL   9398 CD101 216 FYVNVKEQF  79682MLF1IP 217 IYISNSIYF  54967 CXorf48 218 LYSELNKWSF   1591 CYP24A1 219SYLKAVFNL 163720, 199974 CYP4Z2P, CYP4Z1 220 SYSEIKDFL  64421 DCLRE1C221 KYIGNLDLL   8701 DNAH11 222 HYSTLVHMF   8701 DNAH11 223 TFITQSPLL  1767 DNAH5 224 PYFFANQEF  79843 FAM124B 225 TYTNTLERL  55719 FAM178A226 MYLKLVQLF   2175 FANCA 227 IYRFITERF   2301 FOXE3 228 IYQYVADNF  2299 FOXI1 229 IYQFVADSF 344167 FOXI3 230 TYGMVMVTF  84059 GPR98 231AFADVSVKF  84059 GPR98 232 YYLSDSPLL  51512 GTSE1 233 QYLTAAALHNL   3552ILIA 234 SYLPAIWLL   3641 INSL4 235 VYKDSIYYI  84541 KBTBD8 236VYLPKIPSW 157855 KCNU1 237 KYVGQLAVL   9928 KIF14 238 SYLEKVRQL100653049, 3881, KRT31, KRT33A, 3883, 3884, 3885, 3886KRT33B, KRT34, KRT35 239 VYAIFRILL    987 LRBA 240 YYFFVQEKI  84944 MAEL241 SYVKVLHHL 101060230, 4111 MAGEA12 242 VYGEPRELL 392555, 51438 MAGEC2243 SYLELANTL   4163 MCC 244 VHFEDTGKTLLF   4322 MMP13 245 LYPQLFVVL377711, 727957 MROH1 246 KYLSVQLTL 339766 MROH2A 247 SFTKTSPNF 200958MUC20 248 AFPTFSVQL   4588 MUC6 249 RYHPTTCTI   4608 MYBPH 250KYPDIASPTF  89795 NAV3 251 VYTKALSSL  64151 NCAPG 252 AFGQETNVPLNNF  4695 NDUFA2 253 IYGFFNENF  10886 NPFFR2 254 KYLESSATF  91181 NUP210L255 VYQKIILKF 139135 PASD1 256 VFGKSAYLF 118987 PDZD8 257 IFIDNSTQPLHF  5288 PIK3C2G 258 AYAQLGYLL   9033 PKD2L1 259 YFIKSPPSQLF  79949PLEKHS1 260 VYMNVMTRL   5523 PPP2R3A 261 GYIKLINFI  10196 PRMT3 262VYSSQFETI  23362 PSD3 263 RYILENHDF 442247 RFPL4B 264 LYTETRLQF   26150RIBC2 265 SYLNEAFSF 286205 SCAI 266 KYTDVVTEFL  57713 SFMBT2 267SFLNIEKTEILF 347051 SLC10A5 268 IFITKALQI 159371 SLC35G1 269 QYPYLQAFF146857 SLFN13 270 YYSQESKVLYL  55181 SMG8 271 RFLMKSYSF   8435 SOAT2 272RYVFPLPYL   8403 SOX14 273 IYGEKLQFIF  57405 SPC25 274 KQLDIANYELF 51430 SUCO 275 KYGTLDVTF 255928 SYT14 276 QYLDVLHAL  51256 TBC1D7 277FYTFPFQQL   6996 TDG 278 KYVNLVMYF 116238 TLCD1 279 VWLPASVLF  85019TMEM241 280 TYNPNLQDKL   5651 TMPRSS15 281 NYSPGLVSLIL  28677 TRAV9-2282 NYLVDPVTI 129868, 653192 TRIM43, TRIM43B 283 EYQEIFQQL129868, 653192 TRIM43, TRIM43B 284 DYLKDPVTI 391712, 653794TRIM61, TRIM60P14 285 VYVGDALLHAI   7223 TRPC4 286 SYGTILSHI  54986 ULK4287 IYNPNLLTASKF  81839 VANGL1 288 VYPDTVALTF 284403 WDR62 289 FFHEGQYVF389668 XKR9 290 KYGDFKLLEF 143570 XRRA1 291 YYLGSGRETF 152002 XXYLT1 292FYPQIINTF  79776 ZFHX4 293 VYPHFSTTNLI  79776 ZFHX4 294 RFPVQGTVTF 79818 ZNF552 295 SYLVIHERI  84775 ZNF607 296 SYQVIFQHF 344905 ATP13A5297 TYIDTRTVF    827 CAPN6 298 AYKSEVVYF 441402, 728577, 79937CNTNAP3B, CNTNAP3 299 KYQYVLNEF 400823 FAM177B 300 TYPSQLPSL  26290GALNT8 301 KFDDVTMLF   2977 GUCY1A2 302 LYLPVHYGF 253012 HEPACAM2 303LYSVIKEDF 285600 KIAA0825 304 EYNEVANLF  57097 PARP11 305 NYENKQYLF144406 WDR66 306 VYPAEQPQI   2334 AFF2 307 GYAFTLPLF 440138 ALG11 308TFDGHGVFF  29785 CYP2S1 309 KYYRQTLLF  27042 DIEXF 310 IYAPTLLVF  23341DNAJC16 311 EYLQNLNHI  79659 DYNC2H1 312 SYTSVLSRL  57724 EPG5 313KYTHFIQSF  26301 GBGT1 314 RYFKGDYSI   3709 ITPR2 315 FYIPHVPVSF  89866SEC16B 316 VYFEGSDFKF  55164 SHQ1 317 VFDTSIAQLF   6477 SIAH1 318TYSNSAFQYF  28672 TRAV12-3 319 KYSDVKNLI  57623 ZFAT 320 KFILALKVLF  6790 AURKA

TABLE 2A Additional peptides according to the presentinvention, HLA-A*02-binding. SEQ Official ID Gene No. Sequence GeneID(s)Symbol(s) 321 SLWFKPEEL 4831, 654364 NME2, NME1-NME2 322 ALVSGGVAQA64326 RFWD2 323 ILSVVNSQL 80183 KIAA0226L 324 AIFDFCPSV 23268 DNMBP 325RLLPKVQEV 168417, 89958 ZNF679, SAPCD2 326 SLLPLVWKI 1130 LYST 327SIGDIFLKY 1894 ECT2 328 SVDSAPAAV 10635 RAD51AP1 329 FAWEPSFRDQV 1244ABCC2 330 FLWPKEVEL 146206 RLTPR 331 AIWKELISL 55183 RIF1 332 AVTKYTSAK54145, 85236, H2BFS, HIST1H2BK, 8970 HIST1H2BJ 333 GTFLEGVAK 126328NDUFA11 334 GRADALRVL 79713 IGFLR1 335 VLLAAGPSAA 23225 NUP210 336GLMDGSPHFL 157680 VPS13B 337 KVLGKIEKV 987 LRBA 338 LLYDGKLSSA 987 LRBA339 VLGPGPPPL 254359 ZDHHC24 340 SVAKTILKR 55233, 92597 MOB1A, MOB1B

TABLE 2BAdditional peptides according to the present invention, HLA-A*24-binding.SEQ ID Official No. Sequence GeneID(s) Gene Symbol(s) 341 SYLTQHQRI162655, 344065, 9422 ZNF519, ZNF264 342 NYAFLHRTL 200316, 9582 APOBEC3F,APOBEC3B 343 NYLGGTSTI 367 AR 344 EYNSDLHQF 699 BUB1 345 EYNSDLHQFF 699BUB1 346 IYVIPQPHF 57082 CASC5 347 VYAEVNSL 1459 CSNK2A2 348 IYLEHTESI2177 FANCD2 349 QYSIISNVF 28982 FLVCR1 350 KYGNFIDKL 85865 GTPBP10 351IFHEVPLKF 728432, 79664 NARG2 352 QYGGDLTNTF 3673 ITGA2 353 TYGKIDLGF57650 KIAA1524 354 VYNEQIRDLL 81930 KIF18A 355 IYVTGGHLF 113730 KLHDC7B356 NYMPGQLTI 346389 MACC1 357 QFITSTNTF 94025 MUC16 358 YYSEVPVKL 25878MXRA5 359 NYGVLHVTF 204801 NLRP11 360 VFSPDGHLF 143471, 5688PSMA8, PSMA7 361 TYADIGGLDNQI 5700 PSMC1 362 VYNYAEQTL 100526737, 10432,RBM14-RBM4, RBM14, 5936 RBM4 363 SYAELGTTI 23657 SLC7A11 364 KYLNENQLSQL6491 STIL 365 VFIDHPVHL 26011 TENM4 366 QYLELAHSL 4796 TONSL 367LYQDHMQYI 7474 WNT5A 368 KYQNVKHNL 79830 ZMYM1 369 VYTHEVVTL 983 CDK1370 RFIGIPNQF 79659 DYNC2H1 371 AYSHLRYVF 2195 FAT1 372 VYVIEPHSMEF23225 NUP210 373 GYISNGELF 116143, 5534, 5535 WDR92, PPP3R1, PPP3R2 374VFLPRVTEL 5591 PRKDC 375 KYTDYILKI 374462 PTPRQ 376 VYTPVASRQSL 56852RAD18 377 QYTPHSHQF 57521 RPTOR 378 VYIAELEKI 27127 SMC1B 379 VFIAQGYTL160418 TMTC3 380 VYTGIDHHW 25879 DCAF13 381 KYPASSSVF 3217 HOXB7 382AYLPPLQQVF 26523 EIF2C1 383 RYKPGEPITF 163486 DENND1B 384 RYFDVGLHNF55733 HHAT 385 QYIEELQKF 55103 RALGPS2 386 TFSDVEAHF 55609 ZNF280C 387KYTEKLEEI 95681 CEP41 388 IYGEKTYAF 5273 SERPINB10

TABLE 3 Peptides useful for cancer therapies according tothe invention, e.g. personalized cancer therapies. SEQ ID Official  No.Sequence GeneID(s) Gene Symbol(s) 389 EYLPEFLHTF 154664 ABCA13 390RYLWATVTI 259266 ASPM 391 LYQILQGIVF 983 CDK1 392 RYLDSLKAIVF 55839CENPN 393 KYIEAIQWI 81501 DCSTAMP 394 FYQPKIQQF 55215 FANCI 395LYINKANIW 55632 G2E3 396 YYHFIFTTL 2899 GRIK3 397 IYNGKLFDL 11004 KIF2C398 IYNGKLFDLL 11004 KIF2C 399 SYIDVLPEF 4233 MET 400 KYLEKYYNL 4312MMP1 401 VFMKDGFFYF 4312 MMP1 402 VWSDVTPLTF 4320 MMP11 403 TYKYVDINTF4321 MMP12 404 RYLEKFYGL 4321 MMP12 405 NYPKSIHSF 4321 MMP12 406TYSEKTTLF 94025 MUC16 407 VYGIRLEHF 83540 NUF2 408 QYASRFVQL 10733 PLK4409 YFISHVLAF 6241 RRM2 410 RFLSGIINF 83540 NUF2 411 VYIGHTSTI23499, 93035 MACF1, PKHD1L1 412 SYNPLWLRI 259266 ASPM 413 NYLLYVSNF 4486MST1R 414 MYPYIYHVL 54954 FAM120C 415 SYQKVIELF 55872 PBK 416 AYSDGHFLF26011 TENM4 417 VYKVVGNLL 128239 IQGAP3

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, glioblastoma (GB), breastcancer (BRCA), colorectal cancer (CRC), renal cell carcinoma (ROC),chronic lymphocytic leukemia (OLL), hepatocellular carcinoma (HOC),non-small cell and small cell lung cancer (NSCLC, SOLO), Non-Hodgkinlymphoma (NHL), acute myeloid leukemia (AML), ovarian cancer (OC),pancreatic cancer (PC), prostate cancer (PCA), esophageal cancerincluding cancer of the gastric-esophageal junction (OSCAR), gallbladdercancer and cholangiocarcinoma (GBC, CCC), melanoma (MEL), gastric cancer(GO), testis cancer (TO), urinary bladder cancer (UBC), head- and necksquamous cell carcinoma (HNSCC), and uterine cancer (UEC).

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 388. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 295 (see Table 1A, B, C), and their uses inthe immunotherapy of glioblastoma, breast cancer, colorectal cancer,renal cell carcinoma, chronic lymphocytic leukemia, hepatocellularcarcinoma, non-small cell and small cell lung cancer, Non-Hodgkinlymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer,prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, head-and neck squamous cell carcinoma, or uterine cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 70, 80, 323, and 325. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 70, 80, 323, and 325, and their uses in the immunotherapy ofglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer, testis cancer,urinary bladder cancer, head- and neck squamous cell carcinoma, oruterine cancer.

Also preferred are the peptides—alone or in combination—according to thepresent invention selected from the group consisting of SEQ ID NO: 391,and 403. More preferred are the peptides—alone or incombination—selected from the group consisting of SEQ ID NO: 391, and403, and their uses in the immunotherapy of glioblastoma, breast cancer,colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, head-and neck squamous cell carcinoma, or uterine cancer.

As shown in Example 1, many of the peptides according to the presentinvention are found on various tumor types and can, thus, also be usedin the immunotherapy of a variety of indications. Over-expression of theunderlying polypeptides in a variety of cancers, as shown in Example 2,hints towards the usefulness of these peptides in various otheroncological indications.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer, testis cancer,urinary bladder cancer, head- and neck squamous cell carcinoma, oruterine cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 388.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 48 andhas been synthetically produced (e.g. synthesized) as a pharmaceuticallyacceptable salt. Methods to synthetically produce peptides are wellknown in the art. The salts of the peptides according to the presentinvention differ substantially from the peptides in their state(s) invivo, as the peptides as generated in vivo are no salts. The non-naturalsalt form of the peptide mediates the solubility of the peptide, inparticular in the context of pharmaceutical compositions comprising thepeptides, e.g. the peptide vaccines as disclosed herein. A sufficientand at least substantial solubility of the peptide(s) is required inorder to efficiently provide the peptides to the subject to be treated.Preferably, the salts are pharmaceutically acceptable salts of thepeptides. These salts according to the invention include alkaline andearth alkaline salts such as salts of the Hofmeister series comprisingas anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, C⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, SCN⁻ andas cations NH4⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄, (NH₄)₂HPO₄,(NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃, NH₄ClO₄, NH₄I,NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br,Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄, K₂SO₄, KCH₃COO,KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄, NaH₂PO₄, Na₂SO₄,NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, Nal, NaSCN, ZnCl₂ CS₃PO₄, Cs₂HPO₄,CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CSClO₄, CsI, CsSCN,Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr, LiNO₃, LiClO₄,LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂,MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄),Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂, Ca(NO₃)₂,Ca(ClO₄)₂, Ca₁₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄,Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, Bal₂, and Ba(SCN)₂.Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCl, NaCl,and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrine, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 388, preferably containing SEQ IDNo. 1 to SEQ ID No. 295, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are glioblastoma, breast cancer,colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, headand neck squamous cell carcinoma, or uterine cancer.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”,that can be used in the diagnosis of cancer, preferably of glioblastoma,breast cancer, colorectal cancer, renal cell carcinoma, chroniclymphocytic leukemia, hepatocellular carcinoma, non-small cell and smallcell lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovariancancer, pancreatic cancer, prostate cancer, esophageal cancer includingcancer of the gastric-esophageal junction, gallbladder cancer andcholangiocarcinoma, melanoma, gastric cancer, testis cancer, urinarybladder cancer, head and neck squamous cell carcinoma, or uterinecancer. The marker can be over-presentation of the peptide(s)themselves, or over-expression of the corresponding gene(s). The markersmay also be used to predict the probability of success of a treatment,preferably an immunotherapy, and most preferred an immunotherapytargeting the same target that is identified by the biomarker. Forexample, an antibody or soluble TCR can be used to stain sections of thetumor to detect the presence of a peptide of interest in complex withMHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Both therapeutic and diagnostic uses against additional cancerousdiseases are disclosed in the following description of the underlyingexpression products (polypeptides) of the peptides according to theinvention.

A4GNT is frequently expressed in pancreatic cancer cells but notperipheral blood cells and quantitative analysis of A4GNT mRNA expressedin the mononuclear cell fraction of peripheral blood will contribute tothe detection of pancreatic cancer (Ishizone et al., 2006). A4GNT mRNAwas detectable in 80% of patients with an early stage of gastric cancerwhen the cancer cells were limited to the gastric mucosa, and theexpression levels of A4GNT mRNA were increased in association with tumorprogression (Shimizu et al., 2003).

The up-regulated expression of ABCC2 in primary fallopian tubecarcinomas is associated with poor prognosis (Halon et al., 2013).

In human cancer ADAM10 is up-regulated, with levels generallycorrelating with parameters of tumor progression and poor outcome. Inpreclinical models, a selective inhibitor against ADAM10 has been shownto synergize with existing therapies in decreasing tumor growth (Duffyet al., 2009).

AHCYL2 was shown to be down-regulated in colon carcinoma and in a subsetof lung carcinomas (Lleonart et al., 2006). mRNA expression of AHCYL2was described as being potentially associated with the control of p53function as well as the ras-MAPK pathway, methylation andtranscriptional cellular programs, and AHCYL2 may thus be a regulatorysuppressor gene involved in human colon and lung tumors (Lleonart etal., 2006).

AKR1C1 plays a role in cisplatin resistance in cervical, ovarian andlung cancer cells which includes mitochondrial membrane depolarization,ROS production and activation of the JNK pathway (Chen et al., 2015).Significantly higher intratumoral levels of AKR1C1 were found inresponders to neoadjuvant chemotherapy compared with nonresponders(Hlavac et al., 2014).

Expression of AKR1C3 was shown to be positively correlated with anelevated Gleason score in prostate cancer, indicating that AKR1C3 canserve as a promising biomarker for the progression of prostate cancer(Tian et al., 2014). AKR1C3 was shown to catalyze the reduction of4-androstene-3,17-dione to testosterone and estrone to 17β-estradiol,which promotes the proliferation of hormone dependent prostate andbreast cancers, respectively (Byrns et al., 2011). AKR1C3 was shown tobe up-regulated in breast cancer, prostate cancer and skin squamous cellcarcinoma (Byrns et al., 2011; Mantel et al., 2014). AKR1C3 was shown tobe a marker within a gene signature which is able to discriminateresponder patients from non-responders upon chemo-radiotherapy treatmentof patients with locally advanced rectal cancer (Agostini et al., 2015).AKR1C3 was shown to be associated with doxorubicin resistance in humanbreast cancer by activation of anti-apoptosis PTEN/Akt pathway via lossof the tumor suppressor PTEN (Zhong et al., 2015). AKR1C3 was shown tobe associated with a higher risk of lung cancer among people from aChinese county who were exposed to coal emissions (Li et al., 2015a).AKR1C3 was described as a potential therapeutic marker forchoriocarcinoma which is also associated with the development ofmethotrexate resistance in this disease (Zhao et al., 2014a).

The expression of ALDH1 L1 was shown to be down-regulated in HCC andgliomas. The down-regulation of ALDH1 L1 in those cancers was associatedwith poorer prognosis and more aggressive phenotype (Chen et al., 2012;Rodriguez et al., 2008).

ALOX15 is present at high levels in prostate cancer (PCa), lung cancer,breast cancer, melanomas, and colonic adenocarcinomas when compared withnormal tissues (Kelavkar et al., 2002). ALOX15 enzyme activitycontributes to PCa initiation and progression (Kelavkar et al., 2007).

AR has been implicated in the development of various cancers such asprostate, castrate-resistant prostate, breast, glioblastoma multiforme,colon and gastric (Wang et al., 2009b; Yu et al., 2015b; Mehta et al.,2015; Wang et al., 2015a; Sukocheva et al., 2015). In addition topromoting prostate cancer proliferation, androgen signaling through ARleads to apoptosis via inducing the expression of p21 (WAF1/CIP1), acyclin-dependent kinase inhibitor (Yeh et al., 2000).

Mutations in ARMC5 cause macronodular cortisol-producing neoplasias,bilateral macronodular hyperplasias, primary macronodular adrenalhyperplasia, and meningioma (Espiard and Bertherat, 2015; Kirschner andStratakis, 2016; Elbelt et al., 2015).

Pharmacogenomic studies reveal correlations between ATAD5 and anticanceragents (Abaan et al., 2013). ATAD5 is significantly up-regulated inmalignant peripheral nerve sheath tumors (Pasmant et al., 2011).Hepatitis B virus protein X significantly enhances the expression ofATAD5 in HBV-associated hepatocellular carcinoma (Ghosh et al., 2016).Loss of ATAD5 is embryonically lethal in mice, it acts as tumorsuppressor in both mice and humans, and it interacts with components ofthe human Fanconi Anemia pathway. Furthermore, it may be responsible forsome of the phenotypes associated with neurofibromatosis, a hereditarydisease with high risk of tumor growth (Gazy et al., 2015; Jenne et al.,2001). Variants of the ATAD5 gene locus are associated with epithelialovarian cancer risk (Kuchenbaecker et al., 2015). ATAD5 has a bearing onat least one mammalian phenotype of non-small cell lung cancer (Li etal., 2014).

The expression of ATP10B is de-regulated in highly invasive glioma cellsand associated with the invasive behavior (Tatenhorst et al., 2004).

ATP8B4 may be a prognostic marker and therapeutic target in multiplemyeloma patients and other entities (US Patent No. 20070237770 A1) (Niet al., 2012).

ATR encodes ATR serine/threonine kinase, which belongs to thePI3/P14-kinase family. Copy number gain, amplification, or translocationof the ATR gene were observed in oral squamous cell carcinoma cell lines(Parikh et al., 2014). It has been demonstrated that truncating ATRmutations in endometrial cancers are associated with reduceddisease-free and overall survival (Zighelboim et al., 2009).

B3GNT5 is over-expressed in acute myeloid leukemia, and mouse embryonalcarcinoma and its expression is inversely correlated with promotormethylation in glioblastoma (Ogasawara et al., 2011; Etcheverry et al.,2010; Wang et al., 2012). Down-regulation of B3GNT5 through miRNA-203may contribute to the malignancy of hypopharyngeal squamous cellcarcinoma (Wang et al., 2015g). B3GNT5 is associated with breast cancerpatient survival (Potapenko et al., 2015).

Bub1 expression is increased in subsets of lymphomas, breast, gastricand prostate cancers. Bub1 over-expression correlates with poor clinicalprognosis (Ricke and van Deursen, 2011). Bub1 mutations can be found incolorectal carcinomas exhibiting chromosomal instability (Williams etal., 2007).

C4A has been described as a biomarker for polycystic ovary syndrome andendometrial cancer and experimental data suggest that C4 can mediatecancer growth (Galazis et al., 2013; Rutkowski et al., 2010).

In the acute myelomonocytic leukemia cell line JIH3 a chromosomedeletion includes C7orf10 (Pan et al., 2012).

C8 is constitutively expressed by the human hepatoma cell line HepG2 andexpression is strongly enhanced after stimulation with the cytokinesIL-6, IFN-gamma and IL-1 beta (Scheurer et al., 1997).

Cancer-testis antigen specific primers can detect CASC5 in glioblastomamultiforme, one of the most malignant and aggressive tumors with verypoor prognosis. CASC5 has specific binding motifs at the N-terminus (forBub1 and BubR1) and at the C-terminus (for Zwint-1 and hMis14/hNsl1).Disruption of this connection may be able to lead to tumorigenesis(Kiyomitsu et al., 2011; Jiang et al., 2014c). CASC5 interacts with thetumor suppressor pRb (Bogdanov and Takimoto, 2008). CASC5 is highlyexpressed in proliferating somatic cells, tumors and healthy humantestis (Bogdanov and Takimoto, 2008; Sasao et al., 2004). CASC5 islinked to cell growth suppression and maturation enhancement and itsdisruption thus may be a key factor for leukemogenesis (Hayette et al.,2000; Bogdanov and Takimoto, 2008; Chinwalla et al., 2003; Kuefer etal., 2003; Yang et al., 2014a).

CCR4 has been described as a prognostic marker in various tumors such asrenal cell carcinoma, head and neck squamous cell carcinoma, gastriccancer, breast cancer, colon cancer and Hodgkin lymphoma (Ishida et al.,2006; Olkhanud et al., 2009; Yang et al., 2011; Tsujikawa et al., 2013;Al-haidari et al., 2013; Liu et al., 2014a). Studies have revealed thatgastric cancer patients with CCR4-positive tumors had significantlypoorer prognosis compared to those with CCR4-negative tumors (Lee etal., 2009a).

CCR8 expression is increased in monocytic and granulocytic myeloid cellsubsets in peripheral blood of patients with urothelial and renalcarcinomas. Up-regulated expression of CCR8 is also detected withinhuman bladder and renal cancer tissues and primarily limited totumor-associated macrophages. The CCL1/CCR8 axis is a component ofcancer-related inflammation and may contribute to immune evasion(Eruslanov et al., 2013).

A single nucleotide polymorphism in CD101 was shown to be associatedwith pancreatic cancer risk, but results could not be replicated in aprostate cancer case-control and cohort population, thus, requiringfuture research in the possible role of CD101 in pancreatic cancer(Reid-Lombardo et al., 2011). CD101 was identified as one gene of a6-gene signature that discriminated chronic phase from blast crisischronic myeloid leukemia using a Bayesian model averaging approach(Oehler et al., 2009).

CD84 was described as a CD antigen which is differentially abundant inprogressive chronic lymphocytic leukemia as compared to slow-progressiveand stable chronic lymphocytic leukemia (Huang et al., 2014). CD84expression was shown to be significantly elevated from the early stagesof chronic lymphocytic leukemia (Binsky-Ehrenreich et al., 2014).

CENPE expression significantly correlated with glioma grade and mightcomplement other parameters for predicting survival time for gliomapatients (Bie et al., 2011). CENPE is up-regulated in chemo-resistantepithelial ovarian tumors compared to chemo-sensitive tumors (Ju et al.,2009). CENPE is up-regulated in invasive and aggressive-invasiveprolactin pituitary tumors (Wierinckx et al., 2007).

CLDN14 was shown to be up-regulated in gastric cancer (Gao et al.,2013). CLDN14 expression was shown to be associated with lymphaticmetastasis in gastric cancer (Gao et al., 2013). CLDN14 was described toplay a role in the regulation of tumor blood vessel integrity andangiogenesis in mice (Baker et al., 2013).

CLDN3 is highly differentially expressed in many human tumors and mayprovide an efficient molecular tool to specifically identify and targetbiologically aggressive human cancer cells as CLDN3 is a high affinityreceptor of Clostridium perfringens enterotoxin (Black et al., 2015).CLDN3 is frequently over-expressed in several neoplasias, includingovarian, breast, pancreatic, and prostate cancers (Morin, 2005). CLDN3was identified as prostate cancer biomarker as it is highly expressed inprostate cancer (Amaro et al., 2014). Decreased expression of CLDN3 isassociated with a poor prognosis and EMT in completely resected squamouscell lung carcinoma (Che et al., 2015). CLDN3 inhibits canceraggressiveness via Wnt-EMT signaling and is a potential prognosticbiomarker for hepatocellular carcinoma (Jiang et al., 2014b).

CLDN4 is highly differentially expressed in many human tumors and mayprovide an efficient molecular tool to specifically identify and targetbiologically aggressive human cancer cells as CLDN4 is a high affinityreceptor of Clostridium perfringens enterotoxin (Black et al., 2015).CLDN4 is frequently over-expressed in several neoplasias includingovarian, breast, pancreatic, and prostate cancers (Morin, 2005). Anantibody against the extracellular domain of CLDN4 providespro-chemotherapeutic effects in bladder cancer (Kuwada et al., 2015).High expression of CLDN4 was associated with the more differentiatedintestinal-type gastric carcinoma and lost in poorly differentiateddiffuse type. Low expression of CLDN4 was related to lymphangiogenesis(Shareef et al., 2015).

CLDN6 expression was shown to be associated with lymph node metastasisand TNM stage in non-small cell lung cancer (Wang et al., 2015f).Furthermore, low expression of CLDN6 was shown to be associated withsignificantly lower survival rates in patients with non-small cell lungcancer (Wang et al., 2015f). Thus, low CLDN6 expression is anindependent prognostic biomarker that indicates worse prognosis inpatients with non-small cell lung cancer (Wang et al., 2015f). CLDN6 wasshown to be down-regulated in cervical carcinoma and gastric cancer(Zhang et al., 2015; Lin et al., 2013). CLDN6 was shown to beup-regulated in BRCA1-related breast cancer and ovarian papillary serouscarcinoma (Wang et al., 2013b; Heerma van Voss et al., 2014). CLDN6 wasdescribed as a tumor suppressor for breast cancer (Zhang et al., 2015).Gain of CLDN6 expression in the cervical carcinoma cell lines HeLa andC33A was shown to suppress cell proliferation, colony formation invitro, and tumor growth in vivo, suggesting that CLDN6 may function as atumor suppressor in cervical carcinoma cells (Zhang et al., 2015). CLDN6may play a positive role in the invasion and metastasis of ovariancancer (Wang et al., 2013b). CLDN6 was shown to be consistentlyexpressed in germ cell tumors and thus is a novel diagnostic marker forprimitive germ cell tumors (Ushiku et al., 2012). CLDN6 expression wasshown to be positive in most tumors of an assessed set of atypicalteratoid/rhabdoid tumors of the central nervous system, with strongCLDN6 positivity being a potential independent prognostic factor foroutcome of the disease (Dufour et al., 2012).

CLDN9 was shown to be up-regulated in the metastatic Lewis lungcarcinoma cell line p-3LL and in tumors derived from these cells and inpituitary oncocytomas (Sharma et al., 2016; Hong et al., 2014).Knock-down of CLDN9 expression in metastatic Lewis lung carcinoma p-3LLcells was shown to result in significantly reduced motility,invasiveness in vitro and metastasis in vivo, whereas transientover-expression in these cells was shown to enhance their motility(Sharma et al., 2016). Thus, CLDN9 may play an essential role inpromoting lung cancer metastasis (Sharma et al., 2016). CLDN9 was shownto be down-regulated in cervical carcinoma tissues (Zhu et al., 2015a).CLDN9 expression was observed to be correlated with lymphatic metastasisof cervical carcinomas (Zhu et al., 2015a). CLDN9 was described as themost significantly altered and up-regulated gene in pituitaryoncocytomas with higher expression levels in invasive compared tonon-invasive oncocytomas (Hong et al., 2014). Thus, CLDN9 may be animportant biomarker for invasive pituitary oncocytomas (Hong et al.,2014). Over-expression of CLDN9 in the gastric adenocarcinoma cell lineAGS was shown to enhance invasive potential, cell migration and theproliferation rate and is thus sufficient to enhance tumorigenicproperties of a gastric adenocarcinoma cell line (Zavala-Zendejas etal., 2011). Strong CLDN9 expression was shown to be associated with ahigher mortality rate in diffuse-type gastric adenocarcinomas comparedto the intestinal type and its detection was described as a usefulprognostic marker in “intestinal-” and “diffuse-type” gastricadenocarcinomas (Rendon-Huerta et al., 2010).

CLEC5A mRNA expression was shown to be significantly lower in primaryacute myeloid leukemia patients samples than in macrophages andgranulocytes from healthy donors (Batliner et al., 2011). CLEC5A wasdescribed as a novel transcriptional target of the tumor suppressor PU.1in monocytes/macrophages and granulocytes (Batliner et al., 2011).

DCP2 was identified as miR-224 target that was differentially expressedmore than 2-fold in methotrexate resistant human colon cancer cells(Mencia et al., 2011).

DCSTAMP expression is increased in papillary thyroid cancer (Lee et al.,2009b; Kim et al., 2010b). Down-regulation of DCSTAMP leads to adecreased colony formation of MCF-7 cells probably because of decreasedproliferation and cell cycle progression as well as increased apoptosis(Zeng et al., 2015). DCSTAMP is over-expressed in peripheralmacrophages, and dendritic cells and myeloma plasma cells show highsusceptibility to DCSTAMP and are able to transdifferentiate toosteoclasts. Malignant plasma cells expressing cancer stem cellphenotype and high metastasizing capability express osteoclast markerswhich activate the beta3 transcriptional pathway resulting in ERK1/2phosphorylation and initiation of bone resorbing activity (Silvestris etal., 2011). Esculetin and parthenolide suppress c-Fos and nuclear factorof activated T cell c1 signaling pathway resulting in suppressed DCSTAMPexpression, a marker gene for osteoclast differentiation (Ihn et al.,2015; Baek et al., 2015; Cicek et al., 2011; Courtial et al., 2012; Kimet al., 2014a).

SNPs in DENND1B were significantly associated with pancreas cancer risk(Cotterchio et al., 2015).

DNAH17, also known as DNEL2, was described as a homologue to atumor-antigen identified in melanoma patients (Ehlken et al., 2004).DNAH17 was described as one of several candidate genes mapped to a smallchromosome interval associated with sporadic breast and ovariantumorigenesis, and esophageal cancer in the autosomal dominant disordershereditary neuralgic amyotrophy and tylosis (Kalikin et al., 1999).

DNAH2 is one of the genes mutated in ≥10% of patients with chronicmyelomonocytic leukemia (Mason et al., 2015). Genes encodingmicrotubule-associated proteins, such as DNAH2, showed a 10% or higherincidence of genetic aberrations in CpG-island methylatorphenotype-positive clear renal cell carcinomas (Arai et al., 2015).

Re-expression of methylation silenced tumor suppressor genes byinhibiting DNMT3B has emerged as an effective strategy against cancer(Singh et al., 2013).

FAM83B mRNA expression was significantly higher in squamous cellcarcinoma than in normal lung or adenocarcinoma and FAM83B therefore isa novel biomarker for diagnosis and prognosis (Okabe et al., 2015).FAM83B was identified as an oncogene involved in activating CRAF/MAPKsignaling and driving epithelial cell transformation. Elevatedexpression is associated with elevated tumor grade and decreased overallsurvival (Cipriano et al., 2014). Elevated FAM83B expression alsoactivates the PI3K/AKT signaling pathway and confers a decreasedsensitivity to PI3K, AKT, and mTOR inhibitors (Cipriano et al., 2013).

Down-regulation or dysfunction of FANCD2 due to genetic mutations hasbeen reported in different cancer types including breast cancer, acutelymphatic leukemia and testicular seminomas and is associated withcancer development. Otherwise also re-expression and up-regulation ofFANCD2 was shown to be associated with tumor progression and metastasisin gliomas and colorectal cancer (Patil et al., 2014; Shen et al.,2015a; Shen et al., 2015b; Ozawa et al., 2010; Rudland et al., 2010;Zhang et al., 2010a; Smetsers et al., 2012). PI3K/mTOR/Akt pathwaypromotes FANCD2 inducing the ATM/Chk2 checkpoint as DNA damage responseand monoubiquitinilated FANCD2 activates the transcription of the tumorsuppressor TAp63 (Shen et al., 2013; Park et al., 2013).

FOXJ1 expression is up-regulated, associated with tumor stage,histologic grade and size and correlated with prognosis in patients withclear cell renal cell carcinoma (Zhu et al., 2015b). Decreased FOXJ1expression was significantly correlated with clinic stage, lymph nodemetastasis, and distant metastasis, and lower FOXJ1 expressionindependently predicted shorter survival time in gastric carcinoma (Wanget al., 2015c). Over-expression of FOXJ1 can promote tumor cellproliferation and cell-cycle transition in hepatocellular carcinoma andis associated with histological grade and poor prognosis (Chen et al.,2013).

High GINS2 transcript level predicts poor prognosis and correlates withhigh histological grade and endocrine therapy resistance through mammarycancer stem cells in breast cancer patients (Zheng et al., 2014). GINS2was reported to be present at a high level in lung adenocarcinoma andassociated with TNM stages (Liu et al., 2013b). GINS2 express abundantlyand abnormally in many malignant solid tumors, such as breast cancer,melanoma and hepatic carcinoma. Further, over-expression of GINS2 couldpromote proliferation of leukemic cell lines (Zhang et al., 2013a).

GPR98 expression is increased in primary neuroendocrine tumors relativeto normal tissue (Sherman et al., 2013). GPR98 was among the genesassociated with survival of glioblastoma multiforme (Sadeque et al.,2012). GPR98 displays a transcript regulated by glucocorticoids whichare used for the treatment of acute lymphoblastic leukemia as they leadto the induction of apoptosis (Rainer et al., 2012).

GTPBP10 is highly correlated with copy number variation, geneexpression, and patient outcome in glioblastoma (Kong et al., 2016).

GTSE1 expression represses apoptotic signaling and confers cisplatinresistance in gastric cancer cells (Subhash et al., 2015). GTSE1 isover-expressed in uterine leiomyosarcoma (ULMS) and participated in cellcycle regulation.

H2BFS was consistently expressed as a significant cluster associatedwith the low-risk acute lymphoblastic leukemia subgroups (Qiu et al.,2003).

HIST1H2BJ was shown to be down-regulated in brain tumors and wasdescribed as potentially useful for developing molecular markers ofdiagnostic or prognostic value (Luna et al., 2015).

Acetylation of HISTH4A might be a potential target to inactivateembryonic kidney cancer (Wilms tumor) (Yan-Fang et al., 2015).

HIST1H4C may act as risk distinguishing factor for the development oftreatment-related myeloid leukemia (Bogni et al., 2006).

HIST1H4F was observed to be hyper-methylated in prostate cancer whichmight also correlate with the aging of the patient (Kitchen et al.,2016).

A high methylation rate of HIST1H4K was observed in high-gradenon-muscle invasive bladder cancer as well as in prostate cancer and istherefore representing a potential biomarker (Payne et al., 2009;Kachakova et al., 2013).

It was shown that HIST1H4L is significantly up-regulated in ERG+prostate carcinomas (Camoes et al., 2012). HIST1H4L encodes thereplication-dependent histone cluster 1, H4I that is a member of thehistone H4 family (RefSeq, 2002).

HIST2H4B was identified as novel protein in key cellular pathogenicpathways in cells infected with a reovirus subtype that is presently inclinical trials as an anti-cancer oncolytic agent (Berard et al., 2015).

HIST4H4 was one of the genes which showed continuous down-regulation ingastric cancer cells after treatment with immune-conjugates composed ofan alpha-emitter and the monoclonal antibody d9MAb that specificallytarget cells expressing mutant d9-E-cadherin (Seidl et al., 2010).Hyper-methylation of other members of the histon H4 family wassignificantly associated with shorter relapse-free survival in stage Inon-small cell lung cancer (Sandoval et al., 2013).

HIVEP1 was identified as cellular gene disrupted by human T-lymphotropicvirus type 1 integration in lymphoma cell lines (Cao et al., 2015).HIVEP1 was associated with the unfavorable 11q deletion and also withthe unfavorable Binet stages B and C in chronic lymphocytic leukemia(Aalto et al., 2001).

HNRNPH2 is up-regulated in different cancer types including pancreatic,liver and gastric cancer (Honore et al., 2004; Zeng et al., 2007).HNRNPH2 is involved in splicing of the beta-deletion transcript ofhTERT, which is highly expressed in cancer cells and competes andthereby inhibits endogenous telomerase activity (Listerman et al.,2013).

HOXD11 is dysregulated in head and neck squamous cell carcinoma showingstrikingly high levels in cell lines and patient tumor samples.Knockdown of HOXD11 reduced invasion (Sharpe et al., 2014). HOXD11 issignificantly up-regulated in oral squamous cell carcinoma (Rodini etal., 2012). HOXD11 is aberrantly methylated in human breast cancers(Miyamoto et al., 2005). The HOXD11 gene is fused to the NUP98 gene inacute myeloid leukemia with t(2;11)(q31;p15) (Taketani et al., 2002).

ICOS acts as a ligand of programmed death-1 (PD-1) on T cells, inducesthe immune escape of cancer cells and also acts as a receptor mediatinganti-apoptotic effects on cancer cells (Yang et al., 2015c). Murinetumor models have provided significant support for the targeting ofmultiple immune checkpoints involving ICOS during tumor growth (Leungand Suh, 2014). ICOS+ cell infiltration correlates with adverse patientprognosis, identifying ICOS as a new target for cancer immunotherapy(Faget et al., 2013). ICOS can enhance the cytotoxic effect ofcytokine-induced killer cells against cholangiocarcinoma both in vitroand in vivo (He et al., 2011).

Intratumoral expression of IFNG was shown to be associated withexpression of MHC Class II molecules and a more aggressive phenotype inhuman melanomas (Brocker et al., 1988). Autocrine IFNG signaling wasshown to enhance experimental metastatic ability of IFNGgene-transfected mammary adenocarcinoma cells, and was attributed toincreased resistance to NK cells (Lollini et al., 1993).

Triple-negative breast cancer has high tumor expression of IGFBP3associated with markers of poor prognosis (Marzec et al., 2015). A novelcell death receptor that binds specifically to IGFBP3 was identified andmight be used in breast cancer treatment (Mohanraj and Oh, 2011). IGFBP3exhibits pro-survival and growth-promoting properties in vitro (Johnsonand Firth, 2014). IGFBP3 is an independent marker of recurrence of theurothelial cell carcinomas (Phe et al., 2009).

IGFBPL1 is a regulator of insulin-growth factors and is down-regulatedin breast cancer cell lines by aberrant hypermethylation. Methylation inIGFBPL1 was clearly associated with worse overall survival anddisease-free survival (Smith et al., 2007).

IGFLR1 is mutated in colorectal cancer (Donnard et al., 2014). IGFLR1has structural similarity with the tumor necrosis factor receptor family(Lobito et al., 2011).

IL4I1 protein expression is very frequent in tumors. IL4I1 was detectedin tumor-associated macrophages of different tumor entities, inneoplastic cells from lymphomas and in rare cases of solid cancersmainly mesothelioma (Carbonnelle-Puscian et al., 2009). IL4I1up-regulation in human Th17 cells limits their T-cell receptor(TCR)-mediated expansion by blocking the molecular pathway involved inthe activation of the IL-2 promoter and by maintaining high levels ofTob1, which impairs entry into the cell cycle (Santarlasci et al.,2014).

IQGAP3 is over-expressed in lung cancer and is associated with tumorcell growth, migration and invasion. Furthermore, it is up-regulated bychromosomal amplification in hepatocellular carcinoma and the expressionof IQGAP3 is increased in p53-mutated colorectal cancer patients withpoor survival (Katkoori et al., 2012; Yang et al., 2014b; Skawran etal., 2008). IQGAP3 is modulating the EGFR/Ras/ERK signaling cascade andinteracts with Rac/Cdc42 (Yang et al., 2014b; Kunimoto et al., 2009).

Elevated levels of ITGA2 were found in the highly invasive andmetastatic melanoma cell lines compared with normal cultured melanocytesand non-metastatic melanoma cell lines (van Muijen et al., 1995). Theadhesion molecule ITGA2 was up-regulated by IFN-gamma, TNF-alpha, andIL-1-beta in melanoma cells (Garbe and Krasagakis, 1993). Transfectionof ITGA2 into human rhabdomyosarcoma cells which do not express ITGA2,potentiated the frequency of metastases in various organs (Matsuura etal., 1995).

KCNU1 is located on chromosome 8p in an area that is frequently involvedin complex chromosomal rearrangements in breast cancer (Gelsi-Boyer etal., 2005). KCNU1 is one of the top 25 over-expressed extracellularmembrane proteins in hepatoblastomas of pediatric cancer samples(Orentas et al., 2012).

KIAA0226L is thought to be a tumor suppressor gene and ishyper-methylated in cervical cancer. Re-activation of KIAA0226L leads todecreased cell growth, viability, and colony formation (Huisman et al.,2015; Eijsink et al., 2012; Huisman et al., 2013). The methylationpattern of KIAA0226L can be used to differ between precursor lesions andnormal cervix cancer (Milutin et al., 2015). The methylation pattern ofKIAA0226L cannot be used as specific biomarker for cervical cancer(Sohrabi et al., 2014). Re-activation of KIAA0226L partiallyde-methylates its promotor region and also decreases repressive histonemethylations (Huisman et al., 2013).

KIAA1244 over-expression is one of the important mechanisms causing theactivation of the estrogen/ERalpha signaling pathway in thehormone-related growth of breast cancer cells (Kim et al., 2009).Inhibiting the interaction between KIAA1244 and PHB2 may be a newtherapeutic strategy for the treatment of luminal-type breast cancer(Yoshimaru et al., 2013).

KIAA1524 encodes Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A).A critical role of CIP2A has been shown among other for NSCLC, HCC,HNSCC, bladder, pancreatic, cervical, breast, prostate, ovarian andcolorectal cancers (Ventela et al., 2015; Ma et al., 2014; Guo et al.,2015b; Rincon et al., 2015; Guo et al., 2015c; Wu et al., 2015; Peng etal., 2015; Lei et al., 2014; Liu et al., 2014c; Farrell et al., 2014;Fang et al., 2012; He et al., 2012; Bockelman et al., 2012).

KIF18A was shown to be over-expressed in hepatocellular cancer, whichcorrelated with significantly shorter disease free and overall survival.Thus, KIF18A might be a biomarker for hepatocellular cancer diagnosisand an independent predictor of disease free and overall survival aftersurgical resection (Liao et al., 2014). KIF18A expression isup-regulated in specific subtypes of synovial sarcoma (Przybyl et al.,2014). Estrogen strongly induces KIF18A expression in breast cancer,which is associated with increased proliferation and reduced apoptosis(Zou et al., 2014).

Over-expression of KIF20A was detected in pancreatic ductaladenocarcinoma, melanoma, bladder cancer, non-small cell lung cancer andcholangiocellular carcinoma (Imai et al., 2011; Yamashita et al., 2012;Stangel et al., 2015). Recently, it was reported that patients withpancreatic ductal adenocarcinoma vaccinated with a KIF20A-derivedpeptide exhibited better prognosis compared to the control group(Asahara et al., 2013). In addition, silencing of KIF20A resulted in aninhibition of proliferation, motility, and invasion of pancreatic cancercell lines (Stangel et al., 2015).

High expression of KIF26B in breast cancer associates with poorprognosis (Wang et al., 2013c). KIF26B up-regulation was significantlycorrelated with tumor size analysing CRC tumor tissues and pairedadjacent normal mucosa. KIF26B plays an important role in colorectalcarcinogenesis and functions as a novel prognostic indicator and apotential therapeutic target for CRC (Wang et al., 2015d).

KIF2C was shown to be involved in directional migration and invasion oftumor cells (Ritter et al., 2015). Over-expression of KIF2C was shown tobe associated with lymphatic invasion and lymph node metastasis ingastric and colorectal cancer patients (Ritter et al., 2015). KIF2C wasshown to be up-regulated in oral tongue cancer (Wang et al., 2014a).High expression of KIF2C was shown to be associated with lymph nodemetastasis and tumor staging in squamous cell carcinoma of the oraltongue (Wang et al., 2014a). Silencing of KIF2C was shown to result insuppressed proliferation and migration of the human oral squamous cellcarcinoma cell line Tca8113 (Wang et al., 2014a). Mutation of KIF2C wasdescribed as being associated with colorectal cancer (Kumar et al.,2013).

KIFC1 was shown to be essential for proper spindle assembly, stablepole-focusing and survival of cancer cells independently from number offormed centrosomes (normal or supernumerary centrisomes). KIFC1expression was shown to be up-regulated in breast cancer, particularlyin estrogen receptor negative, progesterone receptor negative and triplenegative breast cancer, and 8 human breast cancer cell lines. Inestrogen receptor-positive breast cancer cells, KIFC1 was one of 19other kinesins whose expression was strongly induced by estrogen. Inbreast cancer, the overexpression of KIFC1 and its nuclear accumulationwas shown to correlate with histological grade and predict poorprogression-free and overall survival. In breast cancer cell lines, theoverexpression of KIFC1 was shown to mediate the resistance todocetaxel. The KIFC1 silencing negatively affected the breast cancercell viability (Zou et al., 2014; Pannu et al., 2015; De et al., 2009;Li et al., 2015b). KIFC1 was shown to be overexpressed in ovarian cancerwhich was associated with tumor aggressiveness, advanced tumor grade andstage. KIFC1 was identified as one of three genes, whose higherexpression in primary NSCLC tumors indicated the higher risk fordevelopment of brain metastasis (Grinberg-Rashi et al., 2009).

KL was described as a tumor suppressor which suppresses the epithelialto mesenchymal transition in cervical cancer and which functions as atumor suppressor in several types of human cancers by inhibitinginsulin/IGF1, p53/p21, and Wnt signaling (Xie et al., 2013; Qureshi etal., 2015). KL was described as an aberrantly expressed gene in a numberof cancers, including breast cancer, lung cancer and hepatocellularcarcinoma (Zhou and Wang, 2015). KL was described to be down-regulatedin pancreatic cancer, hepatocellular carcinoma, and other tumors (Zhouand Wang, 2015). KL was described as a novel biomarker for cancer whosedown-regulation was described to result in promoted proliferation andreduced apoptosis of cancer cells. In this context Wnt/β-cateninsignaling is one of several relevant signaling pathways (Zhou and Wang,2015). A KL gene polymorphism was shown to be associated with increasedrisk of colorectal cancer (Liu et al., 2015).

KLHDC7B is associated with cervical squamous cell carcinoma and is apotential biomarker for cervical squamous cell carcinoma (Guo et al.,2015a).

KLHL genes are responsible for several Mendelian diseases and have beenassociated with cancer (Dhanoa et al., 2013).

Focal expression of KRT31 was observed in invasive onychocytic carcinomaoriginating from nail matrix keratinocytes (Wang et al., 2015e).Pilomatricomas are tumors that emulate the differentiation of matrixcells of the hair follicle, showing cortical differentiation, withsequential over-expression of KRT35 and KRT31 keratins (Battistella etal., 2014).

KRT35 was one of the most frequently and most strongly expressed hairkeratins in pilomatrixomas. Pilomatricomas are tumors that emulate thedifferentiation of matrix cells of the hair follicle, showing corticaldifferentiation, with sequential over-expression of KRT35 and KRT31keratins (Battistella et al., 2014).

Knockdown of LCTL allowed hTERT to immortalize human colonic epithelialcells (Kim et al., 2011).

Researchers have observed that inhibition of LRBA expression by RNAinterference, or by a dominant-negative mutant, resulted in the growthinhibition of cancer cells. These findings imply that deregulatedexpression of LRBA contributes to the altered growth properties of acancer cell (Wang et al., 2004).

LRRC8E is over-expressed in osteosarcoma and neuroblastoma tissues incomparison to normal samples (Orentas et al., 2012).

LTA polymorphisms contributed to the increased risk of cancers (Huang etal., 2013a). Bone resorbing factors like LTA are produced by certainsolid and hematologic cancers and have also been implicated intumour-induced hyper-calcemia (Goni and Tolis, 1993). There is a linkbetween the LTA to LTbetaR signaling axis and cancer (Drutskaya et al.,2010). B-cell-derived lymphotoxin promotes castration-resistant prostatecancer (Ammirante et al., 2010).

MACC1 is over-expressed in many cancer entities including gastric,colorectal, lung and breast cancer and is associated with cancerprogression, metastasis and poor survival of patients (Huang et al.,2013b; Ma et al., 2013a; Stein, 2013; Wang et al., 2015b; Wang et al.,2015h; Ilm et al., 2015). MACC1 promotes carcinogenesis throughtargeting beta-catenin and PI3K/AKT signaling pathways, which leads toan increase of c-Met and beta-catenin and their downstream target genesincluding c-Myc, cyclin D1, caspase9, BAD and MMP9 (Zhen et al., 2014;Yao et al., 2015).

MAGEA10 encodes MAGE family member A10, implicated in some hereditarydisorders, such as dyskeratosis congenital (RefSeq, 2002). By a vaccinedirected against MAGEA10 and two other cancer-testis antigens, all ofwhich are known to be targets of cytotoxic-T-lymphocyte responses, morethan two-thirds of breast cancers would be covered (Taylor et al.,2007). MAGEA10 was expressed in 36.7% of the tumor tissues fromhepatocellular carcinoma patients; however, it was not expressed in thepara-cancer tissues (Chen et al., 2003). MAGEA10 was expressed in 14% of79 lung cancer tissues (Kim et al., 2012).

MAGEB6 was identified as new MAGE gene not expressed in normal tissues,except for testis, and expressed in tumors of different histologicalorigins (Lucas et al., 2000). MAGEB6 was found frequently expressed inhead and neck squamous cell carcinoma and mRNA positivity presentedsignificant associations with recognized clinical features for pooroutcome (Zamuner et al., 2015).

MCC interacts with beta-catenin and re-expression of MCC in colorectalcancer cells specifically inhibits Wnt signaling (Fukuyama et al.,2008). The MCC gene is in close linkage with the adenomatous polyposiscoli gene on chromosome 5, in a region of frequent loss ofheterozygosity (LOH) in colorectal cancer (Kohonen-Corish et al., 2007).LOH of MCC gene could be found in both early and advanced stages ofgastric, lung, esophageal and breast cancers (Wang et al., 1999a;Medeiros et al., 1994).

MET was shown to be up-regulated in dedifferentiated liposarcoma and isassociated with melanocytic tumors, hepatocellular carcinoma, non-smallcell lung cancer, hereditary papillary kidney cancers and gastricadenocarcinomas (Petrini, 2015; Finocchiaro et al., 2015; Steinway etal., 2015; Bill et al., 2015; Yeh et al., 2015).

The expression of MMP10 in oral squamous cell carcinoma was intensiveand in verrucous carcinoma was moderate (Kadeh et al., 2015). MMP10contributes to hepatocarcinogenesis in a novel crosstalk with thestromal derived factor 1/C-X-C chemokine receptor 4 axis(Garcia-Irigoyen et al., 2015). Helicobacter pylori infection promotesthe invasion and metastasis of gastric cancer through increasing theexpression of MMP10 (Jiang et al., 2014a). MMP10 promotes tumorprogression through regulation of angiogenic and apoptotic pathways incervical tumors (Zhang et al., 2014).

The elevated level of preoperative MMP13 was found to associate withtumor progression and poor survival in patients with esophageal squamouscell carcinoma (Jiao et al., 2014). PAI-1, a target gene of miR-143,regulates invasion and lung metastasis via enhancement of MMP13expression and secretion in human osteosarcoma cells, suggesting thatthese molecules could be potential therapeutic target genes forpreventing lung metastasis in osteosarcoma patients (Hirahata et al.,2016). MMP13 is already upregulated in Oral lichen planus (OLP) whichhas been classified as a pre-malignant condition for oral squamous cellcarcinoma (OSCC) (Agha-Hosseini and Mirzaii-Dizgah, 2015). MMP13 plays apotentially unique physiological role in the regeneration ofosteoblast-like cells (Ozeki et al., 2016).

MUC5AC is de-regulated in a variety of cancer types includingcolorectal, gastric, lung and pancreatic cancer. Depletion or lowexpression in colorectal and gastric tumors is associated with a moreaggressive behavior and poor prognosis. Over-expression in lung cancerresults in an increased likelihood of recurrence and metastases(Yonezawa et al., 1999; Kocer et al., 2002; Kim et al., 2014b; Yu etal., 1996).

MUC5B is over-expressed in different cancer entities includingcolorectal, lung and breast cancer and is associated with tumorprogression (Sonora et al., 2006; Valque et al., 2012; Walsh et al.,2013; Nagashio et al., 2015). MUC5B can be repressed under the influenceof methylation and can be up-regulated by ATF-1, c-Myc, NFkappaB, Sp1,CREB, TTF-11 and GCR (Perrais et al., 2001; Van, I et al., 2000).

Genetic variants of MUC6 have been reported to modify the risk ofdeveloping gastric cancer (Resende et al., 2011). In salivary glandtumors the expression patterns of MUC6 appear to be very closelycorrelated with the histopathological tumor type indicating theirpotential use to improve diagnostic accuracy (Mahomed, 2011). Studieshave identified a differential expression of MUC6 in breast cancertissues when compared with the non-neoplastic breast tissues(Mukhopadhyay et al., 2011).

A Chinese study identified MXRA5 as the second most frequently mutatedgene in non-small cell lung cancer (Xiong et al., 2012). In coloncancer, MXRA5 was shown to be over-expressed and might serve as abiomarker for early diagnosis and omental metastasis (Zou et al., 2002;Wang et al., 2013a).

MYB can be converted into an oncogenic transforming protein through afew mutations (Zhou and Ness, 2011). MYB is known as oncogene and isassociated with apoptosis, cell cycle control, cell growth/angiogenesisand cell adhesion by regulating expression of key target genes such ascyclooxygenase-2, Bcl-2, BcIX(L) and c-Myc (Ramsay et al., 2003; Stenmanet al., 2010). The oncogenic fusion protein MYB-NFIB and MYBover-expression are found in adenoid cystic carcinoma of the salivarygland and breast, pediatric diffuse gliomas, acute myeloid leukemia andpancreatic cancer (Wallrapp et al., 1999; Pattabiraman and Gonda, 2013;Nobusawa et al., 2014; Chae et al., 2015; Marchio et al., 2010). By thesynergy between MYB and beta-Catenin during Wnt signaling, MYB isassociated with colon tumorigenesis (Burgess et al., 2011). Since MYB isa direct target of estrogen signaling anti-MYB therapy is considered forER-positive breast tumors (Gonda et al., 2008).

N4BP2 has a potential role in the development of nasopharyngealcarcinoma. There is a statistically relevant difference in two differenthaplotype blocks which correlate with the risk of sporadicnasopharyngeal carcinoma. Furthermore, N4BP2 is over-expressed in thesetumor tissues relative to paired normal tissues (Zheng et al., 2007).

In a multistage, case-only genome-wide association study of 12,518prostate cancer cases, NAALADL2 was identified as a locus associatedwith Gleason score, a pathological measure of disease aggressiveness(Berndt et al., 2015). NAALADL2 is over-expressed in prostate and coloncancer and promotes a pro-migratory and pro-metastatic phenotypeassociated with poor survival (Whitaker et al., 2014).

NCAPG is down-regulated in patients with multiple myeloma, acute myeloidleukemia, and leukemic cells from blood or myeloma cells (Cohen et al.,2014). NCAPG may be a multidrug resistant gene in colorectal cancer (Liet al., 2012a). NCAPG is highly up-regulated in the chromophobe subtypeof human cell carcinoma but not in conventional human renal cellcarcinoma (Kim et al., 2010a). Up-regulation of NCAPG is associated withmelanoma progression (Ryu et al., 2007). NCAPG is associated with uvealmelanoma (Van Ginkel et al., 1998). NCAPG shows variable expression indifferent tumor cells (Jager et al., 2000).

NRK encodes Nik related kinase, a protein kinase required for JNKactivation which may be involved in the induction of actinpolymerization in late embryogenesis (RefSeq, 2002). NRK activates thec-Jun N-terminal kinase signaling pathway and may be involved in theregulation of actin cytoskeletal organization in skeletal muscle cellsthrough cofilin phosphorylation (Nakano et al., 2003).

NUP210 was shown to be a candidate gene carrying polymorphismsassociated with the risk of colorectal cancer (Landi et al., 2012).NUP210 was shown to be up-regulated in cervical cancer and suggested toplay a role in the early phase of tumorigenesis (Rajkumar et al., 2011).

ORC1 was shown to be over-expressed in tumor-derived cell lines and ispredicted to be a biomarker in prostate cancer as well as in leukemia(Struyf et al., 2003; Zimmerman et al., 2013; Young et al., 2014).Through its interaction with histone acetyltranferases such as HBO1,ORC1 exerts oncogenic functions in breast cancer (Wang et al., 2010).

Non-carrier of heterozygous mutations in two SNPs in OSCP1 might be abiomarker for susceptibility for non-viral liver carcinoma (Toda et al.,2014).

OVOL2 induces mesenchymal-epithelial transition resulting in decreasedmetastasis (Roca et al., 2013). OVOL2 inhibits c-Myc and Notch1 (Wellset al., 2009). OVOL2 is hyper-methylated in colorectal cancer resultingin its inability to inhibit Wnt signaling (Ye et al., 2016).Over-expression of OVOL2 decreased cell migration and invasion, reducedmarkers for epithelial-mesenchymal transition, and suppressed metastasis(Ye et al., 2016). OVOL2 is down-regulated in colorectal cancer and isinversely correlated with tumor stage (Ye et al., 2016). OVOL2 isregulated by Wnt signaling pathway (Ye et al., 2016).

OXTR is significantly over-expressed in primary small bowel andpancreatic neuroendocrine tumors, small cell carcinoma of the lung,ovarian carcinoma as well as in prostate cancer, mediating cellmigration and metastasis (Morita et al., 2004; Zhong et al., 2010; Carret al., 2012; Carr et al., 2013; Pequeux et al., 2002). However, OXTR1possesses also an inhibitory effect on proliferation of neoplastic cellsof either epithelial, nervous or bone origin, which is thought to bedependent on the receptor localization on the membrane (Cassoni et al.,2004).

PAPPA represents a metastasis-related gene occurring in a range ofcancer types such as NSCLC and hepatocellular carcinoma, where it ispositively associated to growth (VEGF and IGF-I) and transcriptionfactors (NF-kappaB p50, NF-kappaB p65, HIF-1alpha) (Salim et al., 2013;Iunusova et al., 2013; Engelmann et al., 2015). PAPPA regulates mitoticprogression through modulating the IGF-1 signaling pathway in breastcancer and ovarian cancer cells, where it is predominantly found at theprimary site (Boldt and Conover, 2011; Loddo et al., 2014; Becker etal., 2015; Iunusova et al., 2014).

PGAP1 is down-regulated in the adenocarcinoma cell line AsPC-1 (Yang etal., 2016).

PGR is highly associated with breast cancer initiation and progression,where it activates MAPK and PI3K/AKT pathways as well as the expressionof Growth Factors Receptors (GFR) (Jaiswal et al., 2014; Piasecka etal., 2015). PGR (besides HER and estrogen receptor) acts as aclassification factor helping to distinguish between three differentsubtypes of breast cancer (Safarpour and Tavassoli, 2015).

PLA2G7 has strong influence on lipid metabolism in breast, ovarian,melanoma and prostate cancer cells, where a blockage of the enzyme leadsto impaired cancer pathogenicity (Vainio et al., 2011a; Massoner et al.,2013; Kohnz et al., 2015). PLA2G7 is highly associated with prostatecancer and is therefore representing a potential biomarker for this typeof cancer (Vainio et al., 2011 b).

PPP3R1 is up-regulated in hepatocellular carcinoma cells affecting up to10 different signaling pathways (Zekri et al., 2008).

PRKDC is a frequently mutated gene in endometriosis-associated ovariancancer and breast cancer (Er et al., 2016; Wheler et al., 2015). PRKDCis up-regulated in cancerous tissues compared with normal tissues incolorectal carcinoma. Patients with high PRKDC expression show pooreroverall survival (Sun et al., 2016).

An up-regulated expression of PSMA7T was found in metastatic lungcancer, castration-recurrent prostate cancer (CRPC) as well as inprimary colorectal cancer, where it increases the risk of livermetastasis (Hu et al., 2008; Hu et al., 2009; Romanuik et al., 2010; Caiet al., 2010). It was also shown that the amount of PSMA7T correlateswith the transactivation of the androgen receptor (AR) inandrogen/AR-mediated prostate tumor growth (Ogiso et al., 2002).

It was shown that PSMC1 is able to influence cell growth and istherefore representing a potential anti-cancer target in prostatecancer, multiple myeloma and glioblastoma cells (Dahlman et al., 2012;Kim et al., 2008).

RAD18 is implicated in tumorigenesis due to its well-known function inDNA damage bypass, post-replication repair and homologous recombination(Ting et al., 2010). RAD18 Arg302Gln polymorphism is associated with therisk for colorectal cancer and non-small-cell lung cancer (Kanzaki etal., 2008; Kanzaki et al., 2007). RAD18 mediates resistance to ionizingradiation in human glioma cells and knockdown of RAD18 disruptshomologous recombination-mediated repair, resulting in increasedaccumulation of double strand breaks (Xie et al., 2014). Using melanomatissue microarray, it was shown that nuclear RAD18 expression wasup-regulated in primary and metastatic melanoma compared to dysplasticnevi (Wong et al., 2012).

RAD51AP1 was shown to be associated with radiation exposure papillarythyroid cancer (Handkiewicz-Junak et al., 2016). Amplification ofRAD51AP1 was shown to be correlated with cell immortality and a shortersurvival time in ovarian cancer (Sankaranarayanan et al., 2015).RAD51AP1 was described as commonly over-expressed in tumor cells andtissues and disruption of RAD51AP1 function was suggested to be apromising approach in targeted tumor therapy (Parplys et al., 2014).RAD51AP1 transcription was shown to be directly stimulated by the tumorsuppressor MEN1 (Fang et al., 2013). RAD51AP1 was shown to beup-regulated in intrahepatic cholangiocarcinoma, humanpapillomavirus-positive squamous cell carcinoma of the head and neck andin BRCA1-deficient compared to sporadic breast tumors (Martinez et al.,2007; Martin et al., 2007; Obama et al., 2008). Suppression of RAD51AP1was shown to result in growth suppression in intrahepaticcholangiocarcinoma cells, suggesting its involvement in the developmentand/or progression of intrahepatic cholangiocarcinoma (Obama et al.,2008).

Knock-down of RBM14 was shown to block glioblastoma multiforme re-growthafter irradiation in vivo (Yuan et al., 2014). RBM14 was shown to bedown-regulated in renal cell carcinoma (Kang et al., 2008). RBM14 wasdescribed as a potential tumor suppressor in renal carcinoma whichinhibits G(1)-S transition in human kidney cells and suppressesanchorage-independent growth and xenograft tumor formation in part bydown-regulation of the proto-oncogene c-myc (Kang et al., 2008). RBM14was shown to be involved in the migration-enhancing action of PEA3 andMCF7 human cancer cells (Verreman et al., 2011). The RBM14 gene wasshown to be amplified in a subset of primary human cancers includingnon-small cell lung carcinoma, squamous cell skin carcinoma and lymphoma(Sui et al., 2007).

RBM4 is involved in regulatory splicing mechanisms of pre-messenger RNAsuppressing proliferation and migration of various cancer cells (Lin etal., 2014; Wang et al., 2014c). Dysregulations of BBM4 activity werefound in cervical, breast, lung, colon, ovarian and rectal cancers(Liang et al., 2015; Markus et al., 2016).

Serum RCOR3 levels in liver cancer patients were significantly lowerthan those in the patients with moderate chronic hepatitis B and withmild chronic hepatitis B (Xue et al., 2011).

Down-regulation of RFWD2 is correlated with poor prognosis in gastriccancer (Sawada et al., 2013). RFWD2 directly interacts with p27 and thede-regulation of this interaction is involved in tumorigenesis (Choi etal., 2015b; Choi et al., 2015a; Marine, 2012). Up-regulation of RFWD2 iscorrelated with poor prognosis in bladder cancer, gastric cancer, andtriple-negative breast cancer (Ouyang et al., 2015; Li et al., 2016; Liet al., 2012c).

RIF1 is highly expressed in human breast tumors, encodes ananti-apoptotic factor required for DNA repair and is a potential targetfor cancer treatment (Wang et al., 2009a). The role of RIF1 in themaintenance of genomic integrity has been expanded to include theregulation of chromatin structure, replication timing and intra-S phasecheckpoint (Kumar and Cheok, 2014).

In patients diagnosed with visceral multicentric infantilemyofibromatosis novel homozygous variants in the RLTPR gene wereidentified (Linhares et al., 2014).

RNF24 was shown to be up-regulated in esophageal adenocarcinoma andplays a critical role in the progression of Barrett's esophagus toesophageal adenocarcinoma (Wang et al., 2014b). RNF24 was shown to bedifferentially expressed depending on certain risk factors in oralsquamous cell carcinoma (Cheong et al., 2009).

RPGRIP1L suppresses anchorage-independent growth partly through themitotic checkpoint protein Mad2 and is a candidate tumor suppressor genein human hepatocellular carcinoma (Lin et al., 2009).

Over-expression of Rtl1 in the livers of adult mice resulted in highlypenetrant tumor formation and over-expression of RTL1 was detected in30% of analyzed human hepatocellular carcinoma samples (Riordan et al.,2013). Transcriptional activity of the imprinted gene RTL1 was assessedin a panel of 32 Wilms tumors and a massive over-expression was detectedcompared to normal renal tissue (Hubertus et al., 2011).

SAPCD2 (also called p42.3 or C9orf140) encodes a protein initially foundto be expressed in gastric cancer, but not in normal gastric mucosa (Xuet al., 2007). SAPCD2 is over-expressed in different cancer entitiesincluding colorectal, gastric, hepatocellular and brain cancer and highSAPCD2 levels are associated with tumor progression (Sun et al., 2013;Weng et al., 2014; Wan et al., 2014). The optimal pathway of SAPCD2 genein protein regulatory network in gastric cancer is Ras protein, Raf-1protein, MEK, MAPK kinase, MAPK, tubulin, spindle protein, centromereprotein and tumor (Zhang et al., 2012a; Weng et al., 2014).

Lower expression of SEMA3A was shown to be correlated with shorteroverall survival and had independent prognostic importance in patientswith head and neck squamous cell carcinoma (Wang et al., 2016).Over-expression of SEMA3A was shown to suppress migration, invasion andepithelial-to-mesenchymal transition due in part to the inhibition ofNF-kB and SNA12 in head and neck squamous cell carcinoma cell lines(Wang et al., 2016). Thus, SEMA3A serves as a tumor suppressor in headand neck squamous cell carcinoma and may be a new target for thetreatment of this disease (Wang et al., 2016). SEMA3A expression wasshown to be significantly reverse associated with metastasis inhepatocellular carcinoma (Yan-Chun et al., 2015). SEMA3A was describedas being down-regulated in numerous types of cancer, including prostatecancer, breast cancer, glioma, epithelial ovarian carcinoma and gastriccancer (Jiang et al., 2015a; Tang et al., 2014). Low SEMA3A expressionwas shown to be correlated with poor differentiation, vascular invasion,depth of invasion, lymph node metastasis, distant metastasis, advancedTNM stage and poor prognosis in gastric cancer (Tang et al., 2014).SEMA3A was described as a candidate tumor suppressor and potentialprognostic biomarker in gastric carcinogenesis (Tang et al., 2014).

It was shown that missense variations in the SERPINB10 gene possesstumorigenic features leading to an increased risk of prostate cancer(Shioji et al., 2005). In addition, SERPINB10 expression issignificantly up-regulated in metastatic mammary tumors (Klopfleisch etal., 2010).

Expression level of SLC16A14 is significantly associated withprogression-free survival and presents a novel putative marker for theprogression of epithelial ovarian cancer (Elsnerova et al., 2016).

SLC18A1 was showing lower expression in unfavorable neuroblastoma tumortypes as compared to favorable ones (Wilzen et al., 2009).

SLC25A43 was identified as a regulator of cell cycle progression andproliferation through a putative mitochondrial checkpoint in breastcancer cell lines (Gabrielson et al., 2016). SLC25A43 affects drugefficacy and cell cycle regulation following drug exposure in breastcancer cell lines (Gabrielson and Tina, 2013).

SLC28A3 was shown to be down-regulated in pancreatic ductaladenocarcinoma (Mohelnikova-Duchonova et al., 2013). SLC28A3 isassociated with clinical outcome in metastatic breast cancer treatedwith paclitaxel and gemcitabine chemotherapy, overall survival ingemcitabine treated non-small cell lung cancer and overall survival ingemcitabine-based chemoradiation treated pancreatic adenocarcinoma (Liet al., 2012b; Lee et al., 2014b; Marechal et al., 2009). SLC28A3 isassociated with fludarabine resistance in chronic lymphocytic leukemiaand drug resistance in T-cell leukemia (Karim et al., 2011;Fernandez-Calotti et al., 2012).

SLC2A13 was consistently increased in the sphere-forming cells in theprimary cultures of oral squamous cell carcinoma samples and confocalmicroscopy revealed that SLC2A13-expressing cells were embedded in thelimited areas of tumor tissue as a cluster suggesting that SLC2A13 canbe a potential marker for cancer stem cells (Lee et al., 2011). SLC2A13was identified as gene associated with non-small-cell lung cancerpromotion and progression (Bankovic et al., 2010).

Inhibition of SLC35A1 was shown to reduce cancer cell sialylation anddecrease the metastatic potential of cancer cells (Maggioni et al.,2014).

SLC7A11 was shown to be down-regulated in drug resistant variants of theW1 ovarian cancer cell line and thus might play a role in cancer celldrug resistance (Januchowski et al., 2013). SLC7A11 was described tomodulate tumor microenvironment, leading to a growth advantage forcancer (Savaskan and Eyupoglu, 2010). SLC7A11 was described to beinvolved in neurodegenerative processes in glioma (Savaskan et al.,2015). SLC7A11 was shown to be repressed by p53 in the context offerroptosis, and the p53-SLC7A11 axis was described as preserved in thep53(3KR) mutant, and contributes to its ability to suppresstumorigenesis in the absence of the classical tumor suppressionmechanisms (Jiang et al., 2015b). SLC7A11 was described as thefunctional subunit of system Xc—whose function is increased inaggressive breast cancer cells (Linher-Melville et al., 2015). Highmembrane staining for SLC7A11 in cisplatin-resistant bladder cancer wasshown to be associated with a poorer clinical outcome and SLC7A11inhibition was described as a promising therapeutic approach to thetreatment of this disease (Drayton et al., 2014). SLC7A11 was shown tobe differentially expressed in the human promyelocytic leukemia cellline HL-60 that had been exposed to benzene and its metabolites and thushighlights a potential association of SLC7A11 with leukemogenesis (Sarmaet al., 2011). Disruption of SLC7A11 was described to result in growthinhibition of a variety of carcinomas, including lymphoma, glioma,prostate and breast cancer (Chen et al., 2009). Inhibition of SLC7A11was shown to inhibit cell invasion in the esophageal cancer cell lineKYSE150 in vitro and its experimental metastasis in nude mice and thusestablishes a role of SLC7A11 in tumor metastasis (Chen et al., 2009).

SLCO5A1 is located at the plasma membrane and may contribute tochemoresistance of small cell lung carcinoma by affecting theintracellular transport of drugs (Olszewski-Hamilton et al., 2011).SLCO5A1 is the most prominent organic anion transporting polypeptide inmetastatic small cell lung cancer and the mRNA level of SLCO5A1 ishighly increased in hepatic tumors and breast cancer (Kindla et al.,2011; Wlcek et al., 2011; Brenner et al., 2015). Gene fusions inoropharyngeal squamous cell carcinoma are associated withdown-regulation of SLCO5A1 (Guo et al., 2016).

SP5 was down-regulated after depletion of beta-catenin in colorectalcancer cell lines and is a novel direct downstream target in the Wntsignaling pathway (Takahashi et al., 2005). The over-expression of SP5demonstrated activation of the beta-catenin pathway in rare humanpancreatic neoplasms (Cavard et al., 2009). In human colorectalcarcinoma cells displaying de-regulated Wnt signaling, monensin reducedthe intracellular levels of β-catenin leading to a decrease in theexpression of Wnt signaling target genes such as SP5 and a decreasedcell proliferation rate (Tumova et al., 2014).

STIL is among the genes with copy number alterations and copy-neutrallosses of heterozygosity in 15 cortisol-secreting adrenocorticaladenomas (Ronchi et al., 2012). Chromosomal deletions that fuse thisgene and the adjacent locus commonly occur in T cell leukemias, and arethought to arise through illegitimate V-(D)-J recombination events(Karrman et al., 2009; Alonso et al., 2012).

An elevated expression of TBC1 D7 was found in the majority of lungcancers and immunohistochemical staining suggested an association ofTBC1 D7 expression with poor prognosis for NSCLC patients (Sato et al.,2010). Over-expression of TBC7 enhanced ubiquitination of TSC1 andincreased phosphorylation of S6 protein by S6 kinase, that is located inthe mTOR-signaling pathway (Nakashima et al., 2007).

TDG influences the Wnt signaling pathway in an up-regulating manner viainteraction with the transcription factor TCF4 and is thought to be apotential biomarker for colorectal cancer (Xu et al., 2014). On theother hand, a reduced expression of TDG leads to an impaired baseexcision repair (BER) pathway with strong oncogenic features (van deKlundert et al., 2012). A down-regulation of the protein was observed inearly breast cancer esophageal squamous cell carcinoma (ESCC) as well asin gastric cancer (Li et al., 2013; Du et al., 2015; Yang et al.,2015a).

Among the four most frequently mutated genes was TENM4 showingprotein-changing mutations in primary CNS lymphomas (Vater et al.,2015). MDA-MB-175 cell line contains a chromosomal translocation thatleads to the fusion of TENM4 and receptors of the ErbB family. Chimericgenes were also found in neuroblastomas (Wang et al., 1999b; Boeva etal., 2013).

TET2 is a critical regulator for hematopoietic stem cell homeostasiswhose functional impairment leads to hematological malignancies(Nakajima and Kunimoto, 2014). TET2 mutations have an adverse impact onprognosis and may help to justify risk-adapted therapeutic strategiesfor patients with acute myeloid leukemia (Liu et al., 2014b). Nuclearlocalization of TET2 was lost in a significant portion of colorectalcancer tissues, in association with metastasis (Huang et al., 2016).

TKTL1 is associated with the development and progression of multipletumor types such as esophageal squamous cell carcinoma, oral squamouscell carcinoma, lung cancer, colorectal cancer and non-small cell lungcancer (Kayser et al., 2011; Bentz et al., 2013; Grimm et al., 2014).

TMEM67 functions in centriole migration to the apical membrane andformation of the primary cilium. Defects in this gene are a cause ofMeckel syndrome type 3 (MKS3) and Joubert syndrome type 6 (JBTS6)(RefSeq, 2002). TMEM67 is involved in cilia formation and defectivecilia may cause ocular coloboma, tongue tumors, and medulloblastoma(Yang et al., 2015b; Parisi, 2009).

TONSL is involved in lung and esophageal carcinogenesis by stabilizingthe oncogenic protein MMS22L (Nguyen et al., 2012). Further interactionswere shown between TONSL and BRCA1, which acts as a breast and ovariantumor suppressor (Hill et al., 2014).

TP63 translocation was described as an event in a subset of anaplasticlymphoma kinase-positive anaplastic large cell lymphomas which isassociated with an aggressive course of the disease (Hapgood and Savage,2015). TP63 was described to play a complex role in cancer due to itsinvolvement in epithelial differentiation, cell cycle arrest andapoptosis (Lin et al., 2015). The TP63 isoform TAp63 was described to beover-expressed in hematological malignancies while TP63 missensemutations have been reported in squamous cancers and TP63 translocationsin lymphomas and some lung adenocarcinomas (Orzol et al., 2015).Aberrant splicing resulting in the over-expression of the TP63 isoformDeltaNp63 was described to be frequently found in human cancers such ascutaneous squamous cell carcinoma, where it is likely to favor tumorinitiation and progression (Missero and Antonini, 2014; Inoue and Fry,2014).

TRIM59 promotes proliferation and migration of non-small cell lungcancer cells by up-regulating cell cycle related proteins (Zhan et al.,2015). The putative ubiquitin ligase TRIM59 is up-regulated in gastrictumors compared with non-tumor tissues and levels of TRIM59 correlatewith tumor progression and patient survival times. TRIM59 interacts withP53, promoting its ubiquitination and degradation, and TRIM59 mightpromote gastric carcinogenesis via this mechanism (Zhou et al., 2014).

TRPC4 was found to be up-regulated in lung cancer, ovarian cancer, headand neck cancer, kidney cancer and non-small cell lung cancer (Zhang etal., 2010b; Zeng et al., 2013a; Jiang et al., 2013; Park et al., 2016).

ULBP3 is expressed soluble and membrane bound isoform in many tumorcells. Pediatric acute lymphoblastic leukemia blasts expresssignificantly higher levels of ULBP3 compared to adult blasts (Torelliet al., 2014). Much higher expression levels of ULBP3 were found in theleukemia cell line K562 compared to other leukemia cell lines.Furthermore, it can be found in both leukemia cell lines and primarymalignant leukemic cells (Ma et al., 2013b). The ULBP3 locus ismethylated in colorectal cancer cell lines (Bormann et al., 2011).Increased mRNA levels and surface expression levels of ULBP3 have beendetected in the human lung cancer cell line SW-900 (Park et al., 2011).ULBP3 has a higher surface expression in leukemic cells (Ogbomo et al.,2008). ULBP3 levels in different tumor cell lines correlate with NK cellcytotoxicity, however, ULBP3 seems not to be suitable as biomarker (Wanget al., 2008; Linkov et al., 2009). ULBP3 is not expressed in the humannasopharyngeal carcinoma cell line CNE2 (Mei et al., 2007). ULBP3 isexpressed in ovarian cancer and inversely correlated with patientsurvival (Carlsten et al., 2007; McGilvray et al., 2010). B cellsexpress ULBP3 in non-Hodgkin's lymphoma or it can be found in peripheralblood, bone marrow, or lymph nodes (Catellani et al., 2007). ULBP3 isexpressed in breast cancer, the glioblastoma cell line U251, human braintumors, and in head and neck squamous cell carcinoma (Eisele et al.,2006; Butler et al., 2009; Bryant et al., 2011; de Kruijf et al., 2012).Tumor cells express soluble and surface ULBP3 to regulate NK cellactivity (Mou et al., 2014). ULBP3 is over-expressed in certainepithelial tumors. Furthermore, the ULBP3 level in cancer patient serais elevated compared to healthy donors (Mou et al., 2014).

VPS13B alleles are mutated in small cell lung cancers (Iwakawa et al.,2015). Mutations of VPS13B were observed in gastric and colorectalcancers (An et al., 2012).

Frameshift mutations of VPS13C were found in gastric and colorectalcancers with microsatellite instability (An et al., 2012).

WDR62 expression was significantly increased in gastric cancer tissuesand cell lines and was associated with poor differentiation andprognosis. Further, WDR62 expression was elevated in multidrug resistantcells (Zeng et al., 2013b). WDR62 over-expression is related tocentrosome amplification and may be a novel useful differentiationbiomarker and a potential therapy target for ovarian cancer (Zhang etal., 2013b).

Exosome-bound WDR92 inhibits breast cancer cell invasion by degradingamphiregulin mRNA (Saeki et al., 2013). WDR92 potentiates apoptosisinduced by tumor necrosis factor-alpha and cycloheximide (Ni et al.,2009).

WNT5A belongs to the WNT gene family that consists of structurallyrelated genes which encode secreted signaling proteins. These proteinshave been implicated in oncogenesis and in several developmentalprocesses, including regulation of cell fate and patterning duringembryogenesis. The WNT5A gene encodes a member of the WNT family thatsignals through both the canonical and non-canonical WNT pathways. Thisprotein is a ligand for the seven transmembrane receptor frizzled-5 andthe tyrosine kinase orphan receptor 2. This protein plays an essentialrole in regulating developmental pathways during embryogenesis. Thisprotein may also play a role in oncogenesis (RefSeq, 2002). WNT5A isover-expressed in CRC and had a concordance rate of 76% between theprimary tumor and metastatic site (Lee et al., 2014a). WNT5A isup-regulated and a key regulator of the epithelial-to-mesenchymaltransition and metastasis in human gastric carcinoma cells,nasopharyngeal carcinoma and pancreatic cancer (Kanzawa et al., 2013;Zhu et al., 2014; Bo et al., 2013).

XRN1 is likely involved in a number of regulatory mRNA pathways inastrocytes and astrocytoma cells (Moser et al., 2007). Knockdown of XRN1inhibited androgen receptor expression in prostate cancer cells andplays an important role in miR-204/XRN1 axis in prostate adenocarcinoma(Ding et al., 2015).

Genome-wide association studies identified gene polymorphisms in XXYLT1.It has been proposed that these polymorphisms are susceptibility locifor non-small cell lung cancer development (Zhang et al., 2012b).

ZBTB20 promotes cell proliferation in non-small cell lung cancer throughrepression of Fox01 (Zhao et al., 2014b). ZBTB20 expression is increasedin hepatocellular carcinoma and associated with poor prognosis (Wang etal., 2011c). Polymorphism in ZBTB20 gene is associated with gastriccancer (Song et al., 2013).

ZFHX4 is thought to regulate cell differentiation and its suppression islinked to glioma-free survival (Chudnovsky et al., 2014). Papillarytumors of the pineal region show high expression levels of ZFHX4(Fevre-Montange et al., 2006). ZFHX4 was found to be a basal cellcarcinoma susceptibility locus (Stacey et al., 2015).

ZMYM1 is a major interactor of ZNF131 which acts in estrogen signalingand breast cancer proliferation (Oh and Chung, 2012; Kim et al., 2016).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” shall mean that the specific proliferationand activation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, 13 or 14 amino acidsor longer, and in case of MHC class II peptides (elongated variants ofthe peptides of the invention) they can be as long as 15, 16, 17, 18, 19or 20 or more amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 4 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein preferably bind to A*02, A*24 orclass II alleles, as specified. A vaccine may also include pan-bindingMHC class II peptides. Therefore, the vaccine of the invention can beused to treat cancer in patients that are A*02 positive, whereas noselection for MHC class II allotypes is necessary due to the pan-bindingnature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86%.

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 388 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 388, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 388. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spartial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 388, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation do not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 5 Preferred variants and motifs of the HLA-A*02-peptidesaccording to SEQ ID NO: 2, 4, and 6. Position 1 2 3 4 5 6 7 8 9SEQ ID NO. 4 E L A E I V F K V Variants I L A M I M L M M A A I A L A AA V I V L V V A T I T L T T A Q I Q L Q Q A Position 1 2 3 4 5 6 7 8 9SEQ ID NO 2 A L Y G K L L K L Variants V I A M V M I M M A A V A I A A AV V V I V V A T V T I T T A Q V Q I Q Q A Position 1 2 3 4 5 6 7 8 9SEQ ID NO. 6 F L D P A Q R D L Variants V I A M V M I M M A A V A I A AA V V V I V V A T V T I T T A Q V Q I Q Q A

TABLE 6 Preferred variants and motifs of the HLA-A*24-bindingpeptides according to SEQ ID NO: 98, 114, and 158. Position 1 2 3 4 5 67 8 9 10 11 12 SEQ ID 158 I Y E E T R G V L K V F Variant I L F F I F LPosition 1 2 3 4 5 6 7 8 9 10 11 12 SEQ ID 114 Q Y L D G T W S L VariantI F F F I F F Position 1 2 3 4 5 6 7 8 9 10 11 12 SEQ ID 98 V F P R L HN V L F Variant Y I Y L Y I L

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than four amino acids, preferably to a total length of upto 30 amino acids. This may lead to MHC class II binding peptides.Binding to MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 μM, andmost preferably no more than about 10 μM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 388.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 388 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “li”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich provide information onspecific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.

Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

For the identification of peptides of the present invention, a databaseof RNA expression data (Lonsdale, 2013) from about 3000 normal (healthy)tissue samples was screened for genes with near-absent expression invital organ systems, and low expression in other important organsystems. Then, cancer-associated peptides derived from the proteinproducts of these genes were identified by mass spectrometry using theXPRESIDENT™ platform as described herein.

In order to select over-presented peptides, a presentation profile wascalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from cancer samples (N=377A*02-positive samples from 370 donors, N=204 A*24-positive samples) withthe fragmentation patterns of corresponding synthetic reference peptidesof identical sequences. Since the peptides were directly identified asligands of HLA molecules of primary tumors, these results provide directevidence for the natural processing and presentation of the identifiedpeptides on primary cancer tissue obtained from 574 cancer patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary cancer samples, confirmingtheir presentation on primary glioblastoma, breast cancer, colorectalcancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, oruterine cancer.

TUMAPs as identified on multiple cancer and normal tissues werequantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

Furthermore, the discovery pipeline XPRESIDENT® v2.1 allows the directabsolute quantitation of MHC-, preferably HLA-restricted, peptide levelson cancer or other infected tissues. Briefly, the total cell count wascalculated from the total DNA content of the analyzed tissue sample. Thetotal peptide amount for a TUMAP in a tissue sample was measured bynanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of anisotope-labeled version of the TUMAP, the so-called internal standard.The efficiency of TUMAP isolation was determined by spiking peptide:MHCcomplexes of all selected TUMAPs into the tissue lysate at the earliestpossible point of the TUMAP isolation procedure and their detection bynanoLC-MS/MS following completion of the peptide isolation procedure.The total cell count and the amount of total peptide were calculatedfrom triplicate measurements per tissue sample. The peptide-specificisolation efficiencies were calculated as an average from 10 spikeexperiments each measured as a triplicate (see Example 6 and Table 14).

This combined analysis of RNA expression and mass spectrometry dataresulted in the 417 peptides of the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably glioblastoma, breast cancer, colorectalcancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, headand neck squamous cell carcinoma, and uterine cancer that over- orexclusively present the peptides of the invention. These peptides wereshown by mass spectrometry to be naturally presented by HLA molecules onprimary human cancer samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy cells or tissue derived from the same organ as the tumor, orother normal tissue cells, demonstrating a high degree of tumorassociation of the source genes (see Example 2). Moreover, the peptidesthemselves are strongly over-presented on tumor tissue—“tumor tissue” inrelation to this invention shall mean a sample from a patient sufferingfrom cancer, but not on normal tissues (see, e.g., Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. glioblastoma, breast cancer, colorectalcancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, headand neck squamous cell carcinoma, or uterine cancer cells presenting thederived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3). Furthermore, thepeptides when complexed with the respective MHC can be used for theproduction of antibodies and/or TCRs, in particular sTCRs, according tothe present invention, as well. Respective methods are well known to theperson of skill, and can be found in the respective literature as well.Thus, the peptides of the present invention are useful for generating animmune response in a patient by which tumor cells can be destroyed. Animmune response in a patient can be induced by direct administration ofthe described peptides or suitable precursor substances (e.g. elongatedpeptides, proteins, or nucleic acids encoding these peptides) to thepatient, ideally in combination with an agent enhancing theimmunogenicity (i.e. an adjuvant). The immune response originating fromsuch a therapeutic vaccination can be expected to be highly specificagainst tumor cells because the target peptides of the present inventionare not presented on normal tissues in comparable copy numbers,preventing the risk of undesired autoimmune reactions against normalcells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides capable of binding to TCRs and antibodies whenpresented by an MHC molecule. The present description also relates tonucleic acids, vectors and host cells for expressing TCRs and peptidesof the present description; and methods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, an peptide-HLA molecule complex, which is atleast double that of a TCR comprising the unmutated TCR alpha chainand/or unmutated TCR beta chain. Affinity-enhancement of tumor-specificTCRs, and its exploitation, relies on the existence of a window foroptimal TCR affinities. The existence of such a window is based onobservations that TCRs specific for HLA-A2-restricted pathogens have KDvalues that are generally about 10-fold lower when compared to TCRsspecific for HLA-A2-restricted tumor-associated self-antigens. It is nowknown, although tumor antigens have the potential to be immunogenic,because tumors arise from the individual's own cells only mutatedproteins or proteins with altered translational processing will be seenas foreign by the immune system. Antigens that are upregulated oroverexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain.

Therefore, affinity of TCRs or variants of the present description tothe peptides according to the invention can be enhanced by methods wellknown in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with peptide of interest, incubating PBMCs obtained from thetransgenic mice with tetramer-phycoerythrin (PE), and isolating the highavidity T-cells by fluorescence activated cell sorting (FACS)-Caliburanalysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient. In anotheraspect, to obtain T-cells expressing TCRs of the present description,TCR RNAs are synthesized by techniques known in the art, e.g., in vitrotranscription systems. The in vitro-synthesized TCR RNAs are thenintroduced into primary CD8+ T-cells obtained from healthy donors byelectroporation to re-express tumor specific TCR-alpha and/or TCR-betachains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed. In addition to strong promoters, TCRexpression cassettes of the present description may contain additionalelements that can enhance transgene expression, including a centralpolypurine tract (cPPT), which promotes the nuclear translocation oflentiviral constructs (Follenzi et al., 2000), and the woodchuckhepatitis virus posttranscriptional regulatory element (wPRE), whichincreases the level of transgene expression by increasing RNA stability(Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004).

Modifying the TCR-alpha and TCR-beta gene sequences such that each aminoacid is encoded by the optimal codon for mammalian gene expression, aswell as eliminating mRNA instability motifs or cryptic splice sites, hasbeen shown to significantly enhance TCR-alpha and TCR-beta geneexpression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic, such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 388, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques.

Generally, not all of the hosts will be transformed by the vector.Therefore, it will be necessary to select for transformed host cells.One selection technique involves incorporating into the expressionvector a DNA sequence, with any necessary control elements, that codesfor a selectable trait in the transformed cell, such as antibioticresistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbes and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun”, may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvimmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 388,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bispecific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 388, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 388 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:388 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 388, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 388.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of cancers such asglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer (GC), testiscancer (TC), urinary bladder cancer (UBC), head and neck squamous cellcarcinoma (HNSCC), or uterine cancer (UEC).

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 388 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are solid or hematological tumorcells such as glioblastoma, breast cancer, colorectal cancer, renal cellcarcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,non-small cell and small cell lung cancer, Non-Hodgkin lymphoma, acutemyeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer,esophageal cancer including cancer of the gastric-esophageal junction,gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer,testis cancer, urinary bladder cancer, head and neck squamous cellcarcinoma (HNSCC), or uterine cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of glioblastoma, breast cancer, colorectal cancer, renal cellcarcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,non-small cell and small cell lung cancer, Non-Hodgkin lymphoma, acutemyeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer,esophageal cancer including cancer of the gastric-esophageal junction,gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer,testis cancer, urinary bladder cancer, head and neck squamous cellcarcinoma (HNSCC), or uterine cancer. The present invention also relatesto the use of these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a glioblastoma, breast cancer,colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, headand neck squamous cell carcinoma (HNSCC), or uterine cancer marker(poly)peptide, delivery of a toxin to a cancer cell expressing a cancermarker gene at an increased level, and/or inhibiting the activity of acancer marker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods.

The person of skill will understand that either full lengthglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer, testis cancer,urinary bladder cancer, head and neck squamous cell carcinoma (HNSCC),or uterine cancer marker polypeptides or fragments thereof may be usedto generate the antibodies of the invention. A polypeptide to be usedfor generating an antibody of the invention may be partially or fullypurified from a natural source, or may be produced using recombinant DNAtechniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 388polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the marker polypeptide forabove-mentioned cancers used to generate the antibody according to theinvention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating glioblastoma,breast cancer, colorectal cancer, renal cell carcinoma, chroniclymphocytic leukemia, hepatocellular carcinoma, non-small cell and smallcell lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovariancancer, pancreatic cancer, prostate cancer, esophageal cancer includingcancer of the gastric-esophageal junction, gallbladder cancer andcholangiocarcinoma, melanoma, gastric cancer, testis cancer, urinarybladder cancer, head and neck squamous cell carcinoma (HNSCC), oruterine cancer, the efficacy of the therapeutic antibody can be assessedin various ways well known to the skilled practitioner. For instance,the size, number, and/or distribution of cancer in a subject receivingtreatment may be monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.

Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 388, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al. (Porta et al., 1994) which describes the development ofcowpea mosaic virus as a high-yielding system for the presentation offoreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 388.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengiel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “over-expressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament.

The molecule may be used by itself or combined with other molecule(s) ofthe invention or (a) known molecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from glioblastoma,breast cancer, colorectal cancer, renal cell carcinoma, chroniclymphocytic leukemia, hepatocellular carcinoma, non-small cell and smallcell lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovariancancer, pancreatic cancer, prostate cancer, esophageal cancer includingcancer of the gastric-esophageal junction, gallbladder cancer andcholangiocarcinoma, melanoma, gastric cancer, testis cancer, urinarybladder cancer, or uterine cancer, the medicament of the invention ispreferably used to treat glioblastoma, breast cancer, colorectal cancer,renal cell carcinoma, chronic lymphocytic leukemia, hepatocellularcarcinoma, non-small cell and small cell lung cancer, Non-Hodgkinlymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer,prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, headand neck squamous cell carcinoma (HNSCC), or uterine cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue ofglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer, testis cancer,urinary bladder cancer, head and neck squamous cell carcinoma (HNSCC),or uterine cancer patients with various HLA-A HLA-B and HLA-C alleles.It may contain MHC class I and MHC class II peptides or elongated MHCclass I peptides. In addition to the tumor associated peptides collectedfrom several cancer tissues, the warehouse may contain HLA-A*02 andHLA-A*24 marker peptides. These peptides allow comparison of themagnitude of T-cell immunity induced by TUMAPS in a quantitative mannerand hence allow important conclusion to be drawn on the capacity of thevaccine to elicit anti-tumor responses. Secondly, they function asimportant positive control peptides derived from a “non-self” antigen inthe case that any vaccine-induced T-cell responses to TUMAPs derivedfrom “self” antigens in a patient are not observed. And thirdly, it mayallow conclusions to be drawn, regarding the status of immunocompetenceof the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, glioblastoma, breast cancer,colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, headand neck squamous cell carcinoma (HNSCC), and uterine cancer samplesfrom patients and blood from healthy donors were analyzed in a stepwiseapproach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue comparedwith a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromcancer patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine.

As one example, candidate TUMAPs may be identified in the patient by(a1) comparing expression data from the tumor sample to expression datafrom a sample of normal tissue corresponding to the tissue type of thetumor sample to identify proteins that are over-expressed or aberrantlyexpressed in the tumor sample; and (a2) correlating the expression datawith sequences of MHC ligands bound to MHC class I and/or class IImolecules in the tumor sample to identify MHC ligands derived fromproteins over-expressed or aberrantly expressed by the tumor. As anotherexample, proteins may be identified containing mutations that are uniqueto the tumor sample relative to normal corresponding tissue from theindividual patient, and TUMAPs can be identified that specificallytarget the mutation. For example, the genome of the tumor and ofcorresponding normal tissue can be sequenced by whole genome sequencing:For discovery of non-synonymous mutations in the protein-coding regionsof genes, genomic DNA and RNA are extracted from tumor tissues andnormal non-mutated genomic germline DNA is extracted from peripheralblood mononuclear cells (PBMCs). The applied NGS approach is confined tothe re-sequencing of protein coding regions (exome re-sequencing). Forthis purpose, exonic DNA from human samples is captured usingvendor-supplied target enrichment kits, followed by sequencing with e.g.a HiSeq2000 (Illumina). Additionally, tumor mRNA is sequenced for directquantification of gene expression and validation that mutated genes areexpressed in the patients' tumors.

The resultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from glioblastoma, breast cancer, colorectal cancer,renal cell carcinoma, chronic lymphocytic leukemia, hepatocellularcarcinoma, non-small cell and small cell lung cancer, Non-Hodgkinlymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer,prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, testis cancer, urinary bladder cancer, headand neck squamous cell carcinoma (HNSCC), or uterine cancer samples andsince it was determined that these peptides are not or at lower levelspresent in normal tissues, these peptides can be used to diagnose thepresence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for glioblastoma, breast cancer, colorectal cancer, renal cellcarcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,non-small cell and small cell lung cancer, Non-Hodgkin lymphoma, acutemyeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer,esophageal cancer including cancer of the gastric-esophageal junction,gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer,testis cancer, urinary bladder cancer, head and neck squamous cellcarcinoma (HNSCC), or uterine cancer. Presence of groups of peptides canenable classification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1Z show the over-presentation of various peptides in normaltissues (white bars) and different cancers (black bars). FIG. 1A) Genesymbol: IGFBPL1, Peptide: LLPLLPPLSPSLG (SEQ ID NO: 33)—Tissues fromleft to right: 1 cell line (1 pancreatic), 1 normal tissue (1 thyroidgland), 22 cancer tissues (5 brain cancers, 1 breast cancer, 1 coloncancer, 1 esophageal cancer, 1 gallbladder cancer, 1 liver cancer, 10lung cancers, 1 pancreas cancer, 1 stomach cancer); FIG. 11B) Genesymbol: HIVEP1, Peptide: NYIPVKNGKQF (SEQ ID NO: 103)—Tissues from leftto right: 11 cancer tissues (1 brain cancer, 1 liver cancer, 8 lungcancers, 1 prostate cancer); FIG. 1C) Gene symbol: GET4, Peptide:EYLDRIGQLFF (SEQ ID NO: 131)—Tissues from left to right: 2 normaltissues (1 kidney, 1 lung), 41 cancer tissues (2 brain cancers, 1 kidneycancer, 3 liver cancers, 29 lung cancers, 2 prostate cancers, 4 stomachcancers); FIG. 1D) Gene symbol: N4BP2, Peptide: FYINGQYQF (SEQ ID NO:176)—Tissues from left to right: 1 cell line (1 prostate), 3 normaltissues (1 kidney, 1 pituitary gland, 1 skin), 67 cancer tissues (4brain cancers, 2 liver cancers, 42 lung cancers, 12 prostate cancers, 7stomach cancers). FIGS. 1E) to Z) show the over-presentation of variouspeptides in different cancer tissues compared to normal tissues. Theanalyses included data from more than 440 normal tissue samples, and 526cancer samples. Shown are only samples where the peptide was found to bepresented. FIG. 1E) Gene symbol: AKR1C1, AKR1C3, Peptide: HLYNNEEQV (SEQID NO: 16)—Tissues from left to right: 1 cell line (pancreas), 15 cancertissues (1 bile duct cancer, 1 esophageal cancer, 6 liver cancers, 5lung cancers, 2 urinary bladder cancers); FIG. 1F) Gene symbol:RPS26P39, RPS26P11, RPS26, RPS26P28, RPS26P20, RPS26P15, RPS26P50,RPS26P2, RPS26P25, RPS26P58, Peptide: YVLPKLYVKL (SEQ ID NO: 35)—Tissuesfrom left to right: 1 normal tissue (1 leukocyte sample), 8 cancertissues (1 head-and-neck cancer, 3 leukocytic leukemia cancers, 1myeloid cells cancer, 1 gallbladder cancer, 1 colon cancer, 1 lymph nodecancer); FIG. 1G) Gene symbol: CLDN4, CLDN3, CLDN14, CLDN6, CLDN9,Peptide: SLLALPQDLQA (SEQ ID NO: 40)—Tissues from left to right: 21cancer tissues (1 bile duct cancer, 1 breast cancer, 3 colon cancers, 1rectum cancer, 6 lung cancers, 2 ovarian cancers, 1 prostate cancer, 3urinary bladder cancers, 3 uterus cancers); FIG. 1H) Gene symbol:KLHDC7B, Peptide: VLSPFILTL (SEQ ID NO: 42)—Tissues from left to right:18 cancer tissues (1 leukocytic leukemia cancer, 1 myeloid cells cancer,1 breast cancer, 1 kidney cancer, 6 lung cancers, 3 lymph node cancers,2 ovarian cancers, 2 urinary bladder cancers, 1 uterus cancer); FIG. 1I)Gene symbol: ATR, Peptide: SLLSHVIVA (SEQ ID NO: 53)—Tissues from leftto right: 3 cell lines (1 blood cell, 2 pancreas), 21 cancer tissues (1head-and-neck cancer, 1 bile duct cancer, 2 leukocytic leukemia cancers,1 breast cancer, 2 esophageal cancers, 1 gallbladder cancer, 1 kidneycancer, 1 liver cancer, 2 lung cancers, 4 lymph node cancers, 1 ovariancancer, 3 skin cancers, 1 urinary bladder cancer); FIG. 1J) Gene symbol:PGAP1, Peptide: FITDFYTTV (SEQ ID NO: 66)—Tissues from left to right: 1cell line (skin), 1 normal tissue (1 colon), 13 cancer tissues (1head-and-neck cancer, 6 brain cancers, 1 colon cancer, 1 liver cancer, 2skin cancers, 2 urinary bladder cancers); FIG. 1K) Gene symbol: ZNF679,SAPCD2, Peptide: RLLPKVQEV (SEQ ID NO: 325)—Tissues from left to right:4 cell lines (2 blood cells, 1 kidney, 1 large intestine), 22 cancertissues (1 myeloid cells cancer, 1 breast cancer, 1 esophageal cancer, 4colon cancers, 1 rectum cancer, 10 lung cancers, 2 ovarian cancers, 1stomach cancer, 1 urinary bladder cancer); FIG. 1L) Gene symbol:ZDHHC24, Peptide: VLGPGPPPL (SEQ ID NO: 339)—Tissues from left to right:2 cell lines (1 kidney, 1 pancreas), 19 cancer tissues (4 leukocyticleukemia cancers, 1 myeloid cells cancer, 1 bone marrow cancer, 2 braincancers, 1 liver cancer, 2 lung cancers, 6 lymph node cancers, 1 skincancer, 1 uterus cancer); FIG. 1M) Gene symbol: ORC1, Peptide: VYVQILQKL(SEQ ID NO: 111)—Tissues from left to right: 1 normal tissue (1 liver),32 cancer tissues (2 liver cancers, 24 lung cancers, 6 stomach cancers);FIG. 1N) Gene symbol: RIF1, Peptide: IYSFHTLSF (SEQ ID NO: 113)—Tissuesfrom left to right: 28 cancer tissues (1 prostate, 1 brain cancer, 25lung cancers, 2 stomach cancers); FIG. 1O) Gene symbol: ANKRD5, Peptide:RYLNKSFVL (SEQ ID NO: 115)—Tissues from left to right: 1 normal tissue(1 stomach), 25 cancer tissues (1 brain cancer, 2 liver cancers, 17 lungcancers, 2 prostate cancers, 3 stomach cancers); FIG. 1P) Gene symbol:IGFLR1, Peptide: RYGLPAAWSTF (SEQ ID NO: 121)—Tissues from left toright: 20 cancer tissues (2 liver cancers, 17 lung cancers, 1 stomachcancer); FIG. 1Q) Gene symbol: CCR8, Peptide: VYALKVRTI (SEQ ID NO:145)—Tissues from left to right: 25 cancer tissues (25 lung cancers);FIG. 1R) Gene symbol: CLEC5A, Peptide: SYGTVSQIF (SEQ ID NO:148)—Tissues from left to right: 5 normal tissues (1 liver, 3 lungs, 1pituitary gland), 100 cancer tissues (10 brain cancers, 4 liver cancers,74 lung cancers, 1 prostate cancer, 11 stomach cancers); FIG. 1S) Genesymbol: FOXJ1, Peptide: IYKWITDNF (SEQ ID NO: 155)—Tissues from left toright: 4 normal tissues (4 kidneys), 53 cancer tissues (10 braincancers, 1 liver cancer, 26 lung cancers, 1 prostate cancer, 15 stomachcancers); FIG. 1T) Gene symbol: IFNG, Peptide: KYTSYILAF (SEQ ID NO:162)—Tissues from left to right: 3 cell lines (3 prostates), 4 normaltissues (1 liver, 1 lung, 1 pancreas, 1 stomach), 95 cancer tissues (1kidney cancer, 5 liver cancers, 71 lung cancers, 2 prostate cancers, 16stomach cancers); FIG. 1U) Gene symbol: KLHL11, Peptide: EYFTPLLSGQF(SEQ ID NO: 165)—Tissues from left to right: 10 cancer tissues (10 lungcancers); FIG. 1V) Gene symbol: TMEM189, Peptide: LYSPVPFTL (SEQ ID NO:175)—Tissues from left to right: 42 cancer tissues (4 brain cancers, 1liver cancer, 30 lung cancers, 7 stomach cancers); FIG. 1W) Gene symbol:BUB1, Peptide: EYNSDLHQFF (SEQ ID NO: 345)—Tissues from left to right:13 cancer tissues (3 brain cancers, 10 lung cancers); FIG. 1X) Genesymbol: CASC5, Peptide: IYVIPQPHF (SEQ ID NO: 346)—Tissues from left toright: 21 cancer tissues (3 brain cancers, 1 kidney cancer, 1 livercancer, 14 lung cancers, 2 stomach cancers); FIG. 1Y) Gene symbol:KIF18A, Peptide: VYNEQIRDLL (SEQ ID NO: 354)—Tissues from left to right:13 cancer tissues (1 brain cancer, 11 lung cancers, 1 stomach cancer);and FIG. 1Z) Gene symbol: PSMA8, PSMA7, Peptide: VFSPDGHLF (SEQ ID NO:360)—Tissues from left to right: 33 cancer tissues (4 liver cancers, 27lung cancers, 1 prostate cancer, 1 stomach cancer).

FIGS. 2A-2D show exemplary expression profiles of source genes of thepresent invention that are highly over-expressed or exclusivelyexpressed in different cancers in a panel of normal tissues (white bars)and different cancer samples (black bars). FIG. 2A) Gene symbol:MXRA5—Tissues from left to right: 61 normal tissue samples (6 arteries,1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1 adipose tissue, 1adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2gallbladders, 1 kidney, 6 lymph nodes, 1 pancreas, 1 pituitary gland, 1rectum, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 thymus, 1 thyroid gland, 5 tracheas, 1 urinary bladder, 1breast, 5 ovaries, 3 placentas, 1 prostate, 1 testis, 1 uterus) and 70cancer samples (10 breast cancers, 11 lung cancers, 12 ovary cancers, 11esophageal cancers, 26 pancreas cancers); FIG. 2B) Gene symbol:KIF26B—Tissues from left to right: 61 normal tissue samples (6 arteries,1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1 adipose tissue, 1adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2gallbladders, 1 kidney, 6 lymph nodes, 1 pancreas, 1 pituitary gland, 1rectum, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 thymus, 1 thyroid gland, 5 tracheas, 1 urinary bladder, 1breast, 5 ovaries, 3 placentas, 1 prostate, 1 testis, 1 uterus) and 58cancer samples (10 breast cancers, 11 lung cancers, 11 esophagealcancers, 26 pancreas cancers); FIG. 2C) Gene symbol: IL4I1—Tissues fromleft to right: 61 normal tissue samples (6 arteries, 1 brain, 1 heart, 2livers, 2 lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 5 bonemarrows, 1 cartilage, 1 colon, 1 esophagus, 2 gallbladders, 1 kidney, 6lymph nodes, 1 pancreas, 1 pituitary gland, 1 rectum, 1 skeletal muscle,1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thymus, 1 thyroidgland, 5 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 3 placentas,1 prostate, 1 testis, 1 uterus) and 34 cancer samples (11 lung cancers,12 ovary cancers, 11 esophageal cancers); FIG. 2D) Gene symbol:TP63—Tissues from left to right: 61 normal tissue samples (6 arteries, 1brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1 adipose tissue, 1 adrenalgland, 5 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2gallbladders, 1 kidney, 6 lymph nodes, 1 pancreas, 1 pituitary gland, 1rectum, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 thymus, 1 thyroid gland, 5 tracheas, 1 urinary bladder, 1breast, 5 ovaries, 3 placentas, 1 prostate, 1 testis, 1 uterus) and 11esophageal cancer samples

FIGS. 3A-3B show exemplary immunogenicity data: flow cytometry resultsafter peptide-specific multimer staining.

FIGS. 4A-4C shows exemplary results of peptide-specific in vitro CD8+ Tcell responses of a healthy HLA-A*02+ donor. CD8+ T cells were primedusing artificial APCs coated with anti-CD28 mAb and HLA-A*02 in complexwith SEQ ID NO: 2 peptide (FIG. 4A, left panel), SEQ ID NO: 9 peptide(FIG. 4B, left panel) and SEQ ID NO: 331 peptide (FIG. 4C, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*02/SEQ ID NO: 2 (FIG. 4A), A*02/SEQ ID NO: 9 (FIG. 4B) or A*02/SEQ IDNO: 331 (FIG. 4C). Right panels (FIGS. 4A,4B and 4C) show controlstaining of cells stimulated with irrelevant A*02/peptide complexes.Viable singlet cells were gated for CD8+ lymphocytes. Boolean gateshelped excluding false-positive events detected with multimers specificfor different peptides. Frequencies of specific multimer+ cells amongCD8+ lymphocytes are indicated.

FIGS. 5A-5D shows exemplary results of peptide-specific in vitro CD8+ Tcell responses of a healthy HLA-A*24+ donor. CD8+ T cells were primedusing artificial APCs coated with anti-CD28 mAb and HLA-A*24 in complexwith SEQ ID NO: 99 peptide (FIG. 5A, left panel), SEQ ID NO: 119 peptide(FIG. 5B, left panel), SEQ ID NO: 142 peptide (FIG. 5C, left panel) andSEQ ID NO: 174 peptide (FIG. 5D, left panel), respectively. After threecycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*24/SEQ ID NO: 99 (FIG. 5A),A*24/SEQ ID NO: 119 (B), A*24/SEQ ID NO: 142 (C) or A*24/SEQ ID NO: 174(FIG. 5D). Right panels (FIGS. 5A,5B,5C and 5D) show control staining ofcells stimulated with irrelevant A*24/peptide complexes. Viable singletcells were gated for CD8+ lymphocytes. Boolean gates helped excludingfalse-positive events detected with multimers specific for differentpeptides. Frequencies of specific multimer+ cells among CD8+ lymphocytesare indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained under written informed consents ofall patients had been given before surgery or autopsy. Tissues wereshock-frozen immediately after excision and stored until isolation ofTUMAPs at −70° C. or below.

Peptides were selected if two conditions were true: (1) Its underlyingtranscript(s) and/or exon(s) are expressed at very low levels, i.e. themedian reads per kilobase per million reads (RPKM) was required to beless than two, and the 75% quartile was required to be less than 5 forthe following organs: brain, blood vessel, heart, liver, lung, blood. Inaddition, the median RPKM was required to be less than 10 for thefollowing organs: urinary bladder, salivary gland, stomach, adrenalgland, colon, small intestine, spleen, bone marrow, pancreas, muscle,adipose tissue, skin, esophagus, kidney, thyroid gland, pituitary gland,nerve. (2) The peptide was tumor-associated, i.e. found specifically oron tumors or over-expressed compared to a baseline of normal tissues(cf. Example 1).

Sample numbers for HLA-A*02 TUMAP selection were: for pancreatic cancerN=16, for renal cancer N=20, for colorectal cancer N=28, for esophagealcarcinoma including cancer of the gastric-esophageal junction N=15, forprostate tumors N=35, for hepatocellular carcinoma N=16, for non-smallcell lung cancer N=88, for gastric cancer N=29, for breast cancer N=9,for melanoma N=3, for ovarian cancer N=20, for chronic lymphocyticleukemia N=13 (of 12 donors), for urinary bladder cancer N=5, for testiscancer N=1, for small-cell lung cancer N=18 (of 13 donors), forgallbladder cancer and cholangiocarcinoma N=3, for acute myeloidleukemia N=5 (of 4 donors), for glioblastoma N=40, and for uterinecancer N=5.

Sample numbers for HLA-A*24 TUMAP selection were: for gastric cancerN=44, for prostate tumors N=37, for non-small cell lung cancer N=88, forhepatocellular carcinoma N=15, for renal cancer N=2, for colorectalcancer N=1, and for glioblastoma N=17.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to published protocols(Falk et al., 1991; Seeger et al., 1999) with minor modifications usingthe HLA-A*02-specific antibody BB7.2, the HLA-A, B, -C-specific antibodyW6/32, the HLA class II-specific antibody L243, CNBr-activatedsepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose different cancer samples to a baseline of normaltissue samples. Presentation profiles of exemplary over-presentedpeptides are shown in FIGS. 1A-1Z.

Table 8 (A and B) and 9 (A and B) show the presentation on variouscancer entities for selected peptides, and thus the particular relevanceof the peptides as mentioned for the diagnosis and/or treatment of thecancers as indicated (e.g. peptide SEQ ID No. 1 for urinary bladdercancer, esophageal cancer, including cancer of the gastric-esophagealjunction, hepatocellular carcinoma, non-small cell lung cancer, andpancreatic cancer, peptide SEQ ID No. 2 for renal cancer, esophagealcancer, including cancer of the gastric-esophageal, glioblastoma, . . .etc.).

TABLE 8AOverview of presentation of selected HLA-A*02-binding tumor-associatedpeptides of the present invention across entities. GB = glioblastoma, BRCA = breastcancer, CRC = colorectal cancer, RCC = renal cell carcinoma, CLL = chronic lymphocyticleukemia, HCC = hepatocellular carcinoma, NSCLC = non-small cell lung cancer, SCLC =small cell lung cancer, NHL = non-Hodgkin lymphoma (8 samples), AML = acute myeloidleukemia, OC = ovarian cancer, PC = pancreatic cancer, cIPC = pancreatic cancer celllines, PCA prostate cancer and benign prostate hyperplasia, OSCAR = esophageal cancer,including = cancer of the gastric-esophageal junction, GBC_CCC = gallbladder adenocarcinomaand cholangiocarcinoma, MEL = melanoma, GC = gastric cancer, TC = testis cancer, UBC =urinary bladder cancer, UEC = uterine cancer. SEQ ID No. SequencePeptide Presentation on cancer entities 1 HLYNNEEQVUBC, OSCAR, HCC, NSCLC, cIPC 2 ALYGKLLKLRCC, OSCAR, GB, BRCA, CLL, UBC, HCC, SCLC, NSCLC, CRC, OC, GC, NHL 4ELAEIVFKV CRC, CLL, NSCLC, GB 5 SLFGQEVYC HCC, GB, CRC 6 FLDPAQRDLUBC, NSCLC, GB, AML, cIPC 7 AAAAKVPEV NSCLC, GB, CRC 8 KLGPFLLNAGC, GB, cIPC, NSCLC 9 FLGDYVENL CLL, CRC, UBC 10 KTLDVFNIILCLL, GC, GBC_CCC, OSCAR 11 GVLKVFLENV HCC, NSCLC, GC, OC, OSCAR 12GLIYEETRGV GC, OSCAR, NSCLC, NHL 13 VLRDNIQGINSCLC, GBC_CCC, OC, GC, BRCA, OSCAR,CRC, GB, UEC, CLL, RCC, UBC, HCC, MEL, SCLC, NHL 15 ALGDYVHAC HCC, GC 16PLWGKVFYL GBC_CCC, NSCLC, GB, cIPC, CRC 17 ILHEHHIFL RCC, NSCLC 19TLLPTVLTL RCC, UBC, SCLC 20 ALDGHLYAI RCC, UBC, GC 21 SLYHRVLLYRCC, OSCAR, NSCLC 22 MLSDLTLQL CRC, PCA, RCC, NSCLC, BRCA, GC, SCLC, PC,OSCAR 23 AQTVVVIKA GC 24 FLWNGEDSAL PC, CRC 25 IQADDFRTL GC 26 KVDGVVIQLOSCAR, GC 27 KVFGDLDQV OSCAR, GC 29 TLCNKTFTA PCA, GB 30 TVIDECTRI GC 31ALSDETKNNWE AML, CLL V 32 ILADEAFFSVCLL, PC, GB, UBC, PCA, CRC, SCLC, HCC, RCC,OC, NSCLC, MEL, OSCAR, cIPC, GC, BRCA, NHL 34 LLPKKTESHHKCRC, HCC, NSCLC, GB T 35 YVLPKLYVKL CLL, CRC, NSCLC, GBC_CCC, UBC, OSCAR36 KLYGIEIEV NSCLC, GB, RCC 37 ALINDILGELVKL cIPC, CRC, RCC, HCC 38KMQEDLVTL NSCLC, RCC, OC, GC, GBC_CCC, OSCAR, cIPC,GB, BRCA, PCA, PC, UEC, HCC, CRC, SCLC, NHL 39 ALMAVVSGLOSCAR, GBC_CCC, CLL, BRCA, HCC, UBC, NSCLC, OC, GC, MEL 40 SLLALPQDLQAPCA, NSCLC, CRC, UBC, OC 41 FVLPLVVTLOC, OSCAR, CLL, PCA, SCLC, NSCLC, NHL 42 VLSPFILTLNSCLC, RCC, BRCA, UBC, OC, NHL 43 LLWAGPVTACLL, HCC, CRC, NSCLC, RCC, UBC, NHL, TC 44 GLLWQIIKV CRC, NSCLC, GC 45VLGPTPELV CRC, SCLC, GC, cIPC, PC 46 SLAKHGIVALPC, NSCLC, CRC, RCC, OC, cIPC, PCA, NHL 47 GLYQAQVNL NSCLC, SCLC 48TLDHKPVTV OC, PCA, NSCLC 49 LLDESKLTL UBC, OC, PCA, NSCLC, RCC 50EYALLYHTL CRC, GC 51 LLLDGDFTL SCLC, HCC 52 ELLSSIFFLUBC, NSCLC, CRC, RCC 53 SLLSHVIVA cIPC, RCC, GBC_CCC, UBC, NSCLC 54FINPKGNWLL UBC, NSCLC, cIPC 55 IASAIVNEL BRCA, GC 56 KILDLTRVLCRC, NSCLC 57 VLISSTVRL cIPC, RCC, CLL, NHL 58 ALDDSLTSL cIPC, PC, GB 59ALTKILAEL UBC, NSCLC 60 FLIDTSASM UBC, SCLC, RCC 61 HLPDFVKQLBRCA, CLL, GBC_CCC 62 SLFNQEVQI CLL, NHL 63 TLSSERDFAL NSCLC, GC 66FITDFYTTV GB 67 GVIETVTSL NSCLC, GC, CRC 68 ALYGFFFKI UBC 69 GIYDGILHSIUEC, GB 70 GLFSQHFNL HCC, NSCLC 71 GLITVDIAL SCLC, PCA 72 GMIGFQVLLUBC, OC, CLL, GB, GBC_CCC 74 ILDETLENV UBC, SCLC, NHL 76 ILLDESNFNHFLNSCLC 77 IVLSTIASV cIPC, CRC, PCA 81 VLFLGKLLV CRC, UBC 82 VLLRVLILNSCLC, TC 83 ELLEYLPQL PC, GBC_CCC 84 FLEEEITRV CRC, GC 85 STLDGSLHAVOSCAR, PCA, GC 87 YLTEVFLHVV CLL, NHL 89 YLVAHNLLL RCC 90 GAVAEEVLSSI GB92 LLRGPPVARA cIPC, PC, RCC, UBC, OSCAR 93 SLLTQPIFL RCC, HCC 321SLWFKPEEL GC, BRCA, CLL, PC, GB, UBC, PCA, CRC, SCLC,HCC, MEL, OC, NSCLC, GBC_CCC, OSCAR, cIPC, NHL 322 ALVSGGVAQAGBC_CCC, OSCAR, BRCA, CLL, UBC, HCC, PC, SCLC, NSCLC, CRC, OC, NHL 323ILSVVNSQL CRC, GC, BRCA, OC, CLL, NSCLC, NHL 324 AIFDFCPSVNSCLC, CRC, GC, MEL, GB, OSCAR, CLL 325 RLLPKVQEVOSCAR, NSCLC, CRC, SCLC, OC 326 SLLPLVWKINSCLC, CRC, GB, RCC, MEL, CLL, TC 327 SIGDIFLKY GC, GB, CRC, RCC, NSCLC328 SVDSAPAAV SCLC, OC, PC, OSCAR, RCC, NSCLC, UBC 329 FAWEPSFRDQVSCLC, HCC 330 FLWPKEVEL NSCLC, BRCA, SCLC, OC, CLL, NHL 331 AIWKELISLGB, CRC 332 AVTKYTSAK CLL, NSCLC, MEL, NHL 333 GTFLEGVAK RCC, CLL, HCC334 GRADALRVL BRCA, SCLC, CLL 335 VLLAAGPSAA UBC, CLL, NSCLC, GC, cIPC336 GLMDGSPHFL PC, NSCLC 337 KVLGKIEKV RCC, CRC 339 VLGPGPPPLNSCLC, cIPC, CLL, NHL 340 SVAKTILKR NSCLC, OSCAR

Table 8B shows the presentation on additional cancer entities forselected peptides, and thus the particular relevance of the peptides asmentioned for the diagnosis and/or treatment of the cancers asindicated.

TABLE 8BOverview of presentation of selected HLA-A*02 peptides across entities. GB =glioblastoma, BRCA = breast cancer, CRC = colorectal cancer, RCC = renal cell carcinoma,CLL = chronic lymphocytic leukemia, HCC = hepatocellular carcinoma, NSCLC = non-smallcell lung cancer, SCLC = small cell lung cancer, NHL = non-Hodgkin lymphoma, AML = acutemyeloid leukemia, OC = ovarian cancer, PC = pancreatic cancer, BPH = prostate cancer andbenign prostate hyperplasia, OSCAR = esophageal cancer, including cancer of the gastric-oesophageal junction, GBC_CCC = gallbladder adenocarcinoma and cholangiocarcinoma,MEL = melanoma, GC = gastric cancer, UBC = urinary bladder cancer, UTC = uterine cancer,HNSCC = head and neck squamous cell carcinoma. SEQ ID No. SequencePeptide Presentation on cancer entities 1 HLYNNEEQV GBC_CCC 2 ALYGKLLKLcIPC, UTC, PCA, MEL, AML 3 TLLGKQVTL CLL, NSCLC, NHL, AML 5 SLFGQEVYCGBC_CCC, PCA 6 FLDPAQRDL MEL 7 AAAAKVPEV MEL, HNSCC, NHL 8 KLGPFLLNAUTC, HCC 9 FLGDYVENL UTC, AML, OC, cIPC 12 GLIYEETRGV AML, UTC, HNSCC 13VLRDNIQGI AML, HNSCC 17 ILHEHHIFL UTC 18 YVLNEEDLQKV UTC, NSCLC 19TLLPTVLTL GBC_CCC, BRCA, UTC 22 MLSDLTLQL MEL 24 FLWNGEDSALNSCLC, GC, UTC 28 TLYSMDLMKV HNSCC, RCC 32 ILADEAFFSVHNSCC, UTC, AML, GBC_CCC 33 LLLPLLPPLSPSLMEL, NSCLC, GBC_CCC, GC, cIPC, SCLC, GB, PC, G MCC, CRC, HCC 34LLPKKTESHHKT UTC 35 YVLPKLYVKL HNSCC, NHL, AML 36 KLYGIEIEV UTC 37ALINDILGELVKL MEL, UTC 38 KMQEDLVTL MEL, AML 39 ALMAVVSGLHNSCC, NHL, UTC, AML 40 SLLALPQDLQA GBC_CCC, BRCA, UTC 41 FVLPLVVTLAML, CRC, BRCA, HNSCC, UTC 42 VLSPFILTL AML, CLL, UTC 43 LLWAGPVTA HNSCC45 VLGPTPELV OSCAR, GBC_CCC, BRCA 46 SLAKHGIVALUBC, HNSCC, GB, CLL, MEL, UTC, HCC 47 GLYQAQVNL OSCAR 50 EYALLYHTLGBC_CCC 51 LLLDGDFTL OSCAR 53 SLLSHVIVA HCC, AML, OC, OSCAR, HNSCC, MEL,CLL, NHL, BRCA 54 FINPKGNWLL UTC, HNSCC 55 IASAIVNEL HCC, GBC_CCC 56KILDLTRVL GBC_CCC 57 VLISSTVRL MEL 59 ALTKILAEL HCC 60 FLIDTSASMAML, CLL, BRCA, HNSCC, UTC, NHL 61 HLPDFVKQL MEL, AML 64 GLSSSSYELGBC_CCC, HCC 65 KLDGICWQV GBC_CCC, HCC 66 FITDFYTTVMEL, UBC, HCC, HNSCC, CRC 67 GVIETVTSL AML 70 GLFSQHFNLBRCA, UTC, HNSCC, UBC, AML, OSCAR, cIPC 71 GLITVDIAL AML, MEL, UTC 72GMIGFQVLL HNSCC, AML 73 GVPDTIATL GC 74 ILDETLENV AML, BRCA 75 ILDNVKNLLAML 77 IVLSTIASV AML 78 LLWGHPRVA NSCLC 79 SLVPLQILL HCC 80 TLDEYLTYLHCC 81 VLFLGKLLV HNSCC 86 LLVTSLVVV HCC, GBC_CCC 88 ILLNTEDLASL RCC 91SSLEPQIQPV MEL, CLL 93 SLLTQPIFL GBC_CCC 321 SLWFKPEEL UTC, HNSCC, AML322 ALVSGGVAQA AML, GC, cIPC, UTC, MEL 323 ILSVVNSQLMEL, GBC_CCC, AML, OSCAR 324 AIFDFCPSV BRCA, NHL, UTC, AML, HNSCC 325RLLPKVQEV AML, BRCA, UBC, GC 326 SLLPLVWKI AML 327 SIGDIFLKY MEL, AML328 SVDSAPAAV NHL, BRCA, AML, UTC, CLL, HNSCC, MEL 329 FAWEPSFRDQVGBC_CCC 330 FLWPKEVEL AML 331 AIWKELISL MEL, CLL, NSCLC 333 GTFLEGVAKMEL, NSCLC 334 GRADALRVL MEL, GBC_CCC, AML 335 VLLAAGPSAAAML, CRC, UTC, NHL 336 GLMDGSPHFL MEL 338 LLYDGKLSSA CRC, UBC, OC 339VLGPGPPPL MEL, GB, UTC, AML, HCC 340 SVAKTILKR NHL

TABLE 9AOverview of presentation of selected HLA-A*24-binding tumor-associatedpeptides of the present invention across entities. GB = glioblastoma, HCC =hepatocellular carcinoma, NSCLC = non-small cell lung cancer, PCA = prostatecancer, GC = gastric cancer, CRC = colorectal cancer, RCC = renal cell carcinoma.SEQ ID NO. Sequence ENTITIES  96 LYSPVPFTL HCC, NSCLC, GC, GB  97TYTFLKETF PCA, HCC, NSCLC, GB  98 VFPRLHNVLF HCC, NSCLC, GC  99QYILAVPVL NSCLC, GC, GB, PCA 100 VYIESRIGTSTSF GB, HCC, NSCLC, GC 101IYIPVLPPHL HCC, NSCLC 102 VYPFENFEF GC, NSCLC 103 NYIPVKNGKQFPCA, HCC, NSCLC, GB 104 SYLTWHQQI PCA, HCC, NSCLC 105 IYNETITDLLGC, GB, HCC, NSCLC 106 IYNETVRDLL GC, GB, NSCLC 107 KYFPYLVVIHCC, NSCLC, GC 109 LFITGGQFF HCC, NSCLC, GC 110 SYPKIIEEFGB, HCC, NSCLC, GC 111 VYVQILQKL GC, HCC, NSCLC 112 IYNFVESKL NSCLC, GC113 IYSFHTLSF NSCLC, GC, GB 114 QYLDGTWSL NSCLC, GC, GB 115 RYLNKSFVLNSCLC, GC, GB, PCA, HCC 116 AYVIAVHLF GB, PCA, HCC, NSCLC 117 IYLSDLTYIHCC, NSCLC, GC, PCA 118 KYLNSVQYI HCC, NSCLC, GC, GB, PCA 119 VYRVYVTTFNSCLC, GC 120 GYIEHFSLW HCC, NSCLC, GC 121 RYGLPAAWSTF HCC, NSCLC, GC122 EYQARIPEF NSCLC, GC, GB, PCA, HCC 123 VYTPVLEHL NSCLC, GC, GB, HCC124 TYKDYVDLF GC, RCC, GB, PCA, HCC, NSCLC 125 VFSRDFGLLVFGC, HCC, NSCLC 126 PYDPALGSPSRLF NSCLC, GC, PCA, HCC 127 QYFTGNPLFNSCLC, GC, GB, RCC, PCA 128 VYPFDWQYI GB, PCA, HCC, NSCLC, GC 129KYIDYLMTW NSCLC, GC, GB, PCA, HCC 130 VYAHIYHQHF NSCLC, GC, PCA, HCC 131EYLDRIGQLFF NSCLC, GC, RCC, GB, PCA, HCC 132 RYPALFPVLHCC, NSCLC, GC, GB, PCA 133 KYLEDMKTYF HCC, NSCLC, GC, GB 134 AYIPTPIYFPCA, NSCLC, GB 135 VYEAMVPLF GC, NSCLC 136 IYPEWPVVFF GC 137 EYLHNCSYFGC, PCA, HCC, NSCLC 138 VYNAVSTSF NSCLC, GC 139 IFGIFPNQF PCA, NSCLC 142VYVDDIYVI NSCLC, GC 143 KYIFQLNEI GB, NSCLC 144 VFASLPGFLF NSCLC, GC 145VYALKVRTI NSCLC 147 LYLAFPLAF NSCLC, GC, PCA, HCC 148 SYGTVSQIFPCA, HCC, NSCLC, GC, GB 149 SYGTVSQI HCC, NSCLC, GB 150 IYITRQFVQFPCA, HCC, NSCLC, GB 151 AYISGLDVF HCC, NSCLC, PCA 153 VYVPFGGKSMITFNSCLC 154 VYGVPTPHF GB, NSCLC 155 IYKWITDNF HCC, NSCLC, GC, GB 156YYMELTKLLL NSCLC, GC, HCC 157 DYIPASGFALF NSCLC, GB 158 IYEETRGVLKVFHCC, NSCLC, GC 159 IYEETRGVL HCC, NSCLC 160 RYGDGGSSF PCA, NSCLC, GC 161KYPDIVQQF PCA, HCC, NSCLC, GC 162 KYTSYILAF NSCLC, GC, PCA, HCC 163RYLTISNLQF NSCLC 165 EYFTPLLSGQF NSCLC 166 FYTLPFHLI HCC, NSCLC 168RYLEAALRL NSCLC, GC, GB, PCA, HCC 169 NYITGKGDVF NSCLC, PCA 170QYPFHVPLL GC, PCA, HCC, NSCLC 174 VYEKNGYIYF NSCLC, GB 175 YYTQYSQTIGB, NSCLC 176 FYINGQYQF GB, PCA, HCC, NSCLC, GC 177 VYFKAGLDVFPCA, NSCLC 178 NYSSAVQKF PCA, HCC, NSCLC, GB 179 TYIPVGLGRLL NSCLC, GC180 KYLQVVGMF NSCLC, GB 182 AYAQLGYLLF NSCLC 183 PYLQDVPRI NSCLC, GB 186VFTTSSNIF NSCLC, GB 187 AYAANVHYL NSCLC 188 GYKTFFNEF NSCLC 192RYSTFSEIF HCC, NSCLC, GC 194 VYQSLSNSL NSCLC 195 AYIKGGWILRCC, HCC, NSCLC, GC 196 GYIRGSWQF NSCLC, GC 197 IFTDIFHYLHCC, NSCLC, GC, GB 199 SYLNHLNNL NSCLC 201 GYNPNRVFF GB, NSCLC 202RYVEGIVSL NSCLC 204 EYLSTCSKL NSCLC, HCC 206 NYLDVATFLNSCLC, GC, GB, PCA, HCC 207 LYSDAFKFIVF NSCLC 209 AFIETPIPLF NSCLC 210IYAGVGEFSF NSCLC, GC 215 SYVASFFLL GC, NSCLC 217 IYISNSIYF NSCLC, GC 221KYIGNLDLL NSCLC, GB 223 TFITQSPLL NSCLC 225 TYTNTLERL NSCLC 226MYLKLVQLF HCC, GC 228 IYQYVADNF NSCLC 229 IYQFVADSF NSCLC 232 YYLSDSPLLNSCLC, GC 234 SYLPAIWLL GC 235 VYKDSIYYI GB, PCA, HCC, NSCLC, GC 236VYLPKIPSW HCC, NSCLC 238 SYLEKVRQL NSCLC 240 YYFFVQEKI HCC, NSCLC 243SYLELANTL PCA, NSCLC 248 AFPTFSVQL NSCLC 249 RYHPTTCTI NSCLC 250KYPDIASPTF HCC 251 VYTKALSSL NSCLC, HCC 252 AFGQETNVPLNNF HCC 253IYGFFNENF HCC 254 KYLESSATF NSCLC 255 VYQKIILKF HCC 257 IFIDNSTQPLHF HCC259 YFIKSPPSQLF NSCLC, GC 260 VYMNVMTRL NSCLC 261 GYIKLINFI GC 262VYSSQFETI GB 264 LYTETRLQF NSCLC 265 SYLNEAFSF PCA 266 KYTDVVTEFLHCC, NSCLC, GC, GB, PCA 268 IFITKALQI GC 269 QYPYLQAFF NSCLC 271RFLMKSYSF HCC 274 KQLDIANYELF NSCLC, GB, HCC 275 KYGTLDVTF NSCLC 276QYLDVLHAL GC, RCC 277 FYTFPFQQL GC, RCC, PCA, HCC, NSCLC 280 TYNPNLQDKLHCC 281 NYSPGLVSLIL NSCLC 284 DYLKDPVTI NSCLC 285 VYVGDALLHAI PCA 286SYGTILSHI NSCLC 288 VYPDTVALTF NSCLC, GC 289 FFHEGQYVF GC 290 KYGDFKLLEFPCA, GB 295 SYLVIHERI NSCLC, GC 296 SYQVIFQHF NSCLC, GC 297 TYIDTRTVFPCA, HCC, NSCLC, GC 298 AYKSEVVYF NSCLC, GB 299 KYQYVLNEF NSCLC, GC, GB300 TYPSQLPSL CRC, GC 301 KFDDVTMLF NSCLC, GC, HCC 303 LYSVIKEDFGB, PCA, HCC, NSCLC, GC 304 EYNEVANLF HCC, NSCLC, GC, RCC, GB, PCA 305NYENKQYLF NSCLC, GB, HCC 306 VYPAEQPQI NSCLC 307 GYAFTLPLF NSCLC, GC 308TFDGHGVFF NSCLC, GC 309 KYYRQTLLF PCA, HCC, NSCLC, GC, GB 310 IYAPTLLVFGC, GB, RCC, HCC, NSCLC 311 EYLQNLNHI PCA 312 SYTSVLSRL PCA, HCC, NSCLC313 KYTHFIQSF NSCLC, GC, RCC, GB, PCA, HCC 314 RYFKGDYSI GB, HCC 315FYIPHVPVSF HCC, NSCLC 316 VYFEGSDFKF GB, PCA, HCC, NSCLC 317 VFDTSIAQLFGB, RCC, HCC, NSCLC, GC 318 TYSNSAFQYF GC, RCC, PCA, HCC, NSCLC 319KYSDVKNLI PCA, HCC, NSCLC, GB 320 KFILALKVLF HCC, NSCLC 341 SYLTQHQRIPCA, NSCLC 343 NYLGGTSTI PCA, HCC, GB 344 EYNSDLHQFGB, RCC, HCC, NSCLC, GC 345 EYNSDLHQFF GB, NSCLC 346 IYVIPQPHFNSCLC, GC, GB, HCC 347 VYAEVNSL GB, NSCLC, GC 348 IYLEHTESIGC, HCC, NSCLC 349 QYSIISNVF GC, HCC, NSCLC 350 KYGNFIDKL NSCLC, GC, HCC351 IFHEVPLKF HCC, NSCLC 352 QYGGDLTNTF NSCLC, GB 353 TYGKIDLGFHCC, NSCLC, GC, GB 354 VYNEQIRDLL NSCLC, GC, GB 355 IYVTGGHLFHCC, NSCLC, GC, RCC, GB, PCA 356 NYMPGQLTI RCC, NSCLC, GC 357 QFITSTNTFNSCLC 358 YYSEVPVKL NSCLC, GB 359 NYGVLHVTF RCC, HCC, NSCLC 360VFSPDGHLF GB, PCA, HCC, NSCLC 361 TYADIGGLDNQI PCA, NSCLC, GC, GB, RCC362 VYNYAEQTL GC, GB, NSCLC 363 SYAELGTTI GB, NSCLC, GC 365 VFIDHPVHLNSCLC, GB 366 QYLELAHSL HCC, NSCLC, GC 367 LYQDHMQYIHCC, NSCLC, GC, GB, PCA 368 KYQNVKHNL NSCLC, HCC 369 VYTHEVVTL NSCLC 370RFIGIPNQF PCA 371 AYSHLRYVF GB, PCA, HCC, NSCLC 373 GYISNGELF PCA, HCC375 KYTDYILKI NSCLC 376 VYTPVASRQSL HCC, NSCLC, GC, GB, PCA 377QYTPHSHQF HCC, NSCLC 378 VYIAELEKI HCC, NSCLC 380 VYTGIDHHWNSCLC, GC, RCC, GB, PCA, HCC 382 AYLPPLQQVF PCA, HCC, NSCLC, GC, RCC, GB383 RYKPGEPITF GB, PCA, HCC, NSCLC 384 RYFDVGLHNFGC, GB, PCA, HCC, NSCLC 385 QYIEELQKF NSCLC, HCC 386 TFSDVEAHFHCC, NSCLC, GC, GB 387 KYTEKLEEI HCC, NSCLC, GB, PCA 388 IYGEKTYAFHCC, NSCLC, GC, RCC, GB, PCA 389 EYLPEFLHTF NSCLC 390 RYLWATVTIGC, HCC, NSCLC 391 LYQILQGIVF NSCLC, GC, GB, RCC, HCC 392 RYLDSLKAIVFNSCLC, GC, RCC, HCC 393 KYIEAIQWI HCC, NSCLC 394 FYQPKIQQFGB, PCA, HCC, NSCLC, GC 395 LYINKANIW NSCLC, GC, HCC 396 YYHFIFTTL GB397 IYNGKLFDL GB, NSCLC, GC 398 IYNGKLFDLL CRC, GC, RCC, GB, PCA,HCC, NSCLC 399 SYIDVLPEF HCC, NSCLC, GC, RCC, GB, PCA 400 KYLEKYYNLNSCLC 401 VFMKDGFFYF NSCLC, GC, PCA 402 VWSDVTPLTFNSCLC, CRC, GC, RCC, GB, PCA, HCC 403 TYKYVDINTF NSCLC, GC 404 RYLEKFYGLNSCLC, GC, HCC 405 NYPKSIHSF NSCLC 406 TYSEKTTLF NSCLC, GC 407 VYGIRLEHFHCC, NSCLC, GC, GB 408 QYASRFVQL GC, GB, HCC, NSCLC 409 YFISHVLAFGC, NSCLC 410 RFLSGIINF NSCLC, GC, GB, HCC 411 VYIGHTSTI NSCLC 412SYNPLWLRI GB, RCC, HCC, NSCLC, GC 413 NYLLYVSNF HCC, NSCLC, GC 414MYPYIYHVL HCC, NSCLC, GC, GB, PCA 415 SYQKVIELFPCA, HCC, NSCLC, CRC, GC, RCC, GB 416 AYSDGHFLF NSCLC, GC, RCC, GB, PCA,HCC 417 VYKVVGNLL GB, RCC, HCC, NSCLC, GC

Table 9B show the presentation on additional cancer entities forselected peptides, and thus the particular relevance of the peptides asmentioned for the diagnosis and/or treatment of the cancers asindicated.

TABLE 9BOverview of presentation of selected HLA-A*24 peptides across cancerentities. GB = glioblastoma, CRC = colorectal cancer, RCC = renal cellcarcinoma, HCC = hepatocellular carcinoma, NSCLC = non-small cell lungcancer, PCA = prostate cancer and benign prostate hyperplasia, GC = gastriccancer. SEQ ID No. Sequence Peptide Presentation on cancer entities  50EYALLYHTL PCA, HCC, NSCLC, CRC, GC, RCC 104 SYLTWHQQI GB 108 PYLVVIHTLNSCLC 110 SYPKIIEEF PCA 132 RYPALFPVL RCC 135 VYEAMVPLF HCC 140RYLINSYDF NSCLC 141 SYNGHLTIWF GB 146 NYYERIHAL NSCLC 148 SYGTVSQIF CRC152 KFFDDLGDELLF NSCLC 155 IYKWITDNF PCA 164 HYVPATKVF NSCLC 167RYGFYYVEF GB 171 NYEDHFPLL NSCLC 172 VFIFKGNEF NSCLC 173 QYLEKYYNL NSCLC181 VYPPYLNYL PCA 184 IYSVGAFENF NSCLC 185 QYLVHVNDL GB 189 AYFKQSSVFNSCLC 190 LYSELTETL NSCLC 191 TYPDGTYTGRIF NSCLC 193 LYLENNAQTQF NSCLC198 DYVGFTLKI NSCLC 200 VFIHHLPQF HCC 203 VYNVEVKNAEF NSCLC 204EYLSTCSKL PCA 205 VYPVVLNQI NSCLC 208 TYLEKIDGF NSCLC 211 VFKSEGAYFNSCLC 212 SYAPPSEDLF NSCLC 213 SYAPPSEDLFL NSCLC 214 KYLMELTLI NSCLC 216FYVNVKEQF NSCLC 218 LYSELNKWSF NSCLC 219 SYLKAVFNL NSCLC 220 SYSEIKDFLNSCLC 222 HYSTLVHMF NSCLC 224 PYFFANQEF HCC 227 IYRFITERF NSCLC 230TYGMVMVTF NSCLC 231 AFADVSVKF NSCLC 233 QYLTAAALHNL NSCLC 237 KYVGQLAVLHCC 239 VYAIFRILL GC 241 SYVKVLHHL HCC 242 VYGEPRELL HCC 244VHFEDTGKTLLF NSCLC 245 LYPQLFVVL GC 246 KYLSVQLTL NSCLC 247 SFTKTSPNFHCC 256 VFGKSAYLF NSCLC 258 AYAQLGYLL NSCLC 263 RYILENHDF HCC 267SFLNIEKTEILF HCC 270 YYSQESKVLYL HCC 272 RYVFPLPYL NSCLC 273 IYGEKLQFIFNSCLC 278 KYVNLVMYF NSCLC 279 VWLPASVLF NSCLC 282 NYLVDPVTI NSCLC 283EYQEIFQQL NSCLC 287 IYNPNLLTASKF NSCLC 291 YYLGSGRETFNSCLC, GB, PCA, HCC 292 FYPQIINTF NSCLC 293 VYPHFSTTNLI HCC 294RFPVQGTVTF PCA 302 LYLPVHYGF NSCLC 342 NYAFLHRTL NSCLC 344 EYNSDLHQF PCA348 IYLEHTESI GB 350 KYGNFIDKL PCA 364 KYLNENQLSQL NSCLC 372 VYVIEPHSMEFNSCLC 374 VFLPRVTEL NSCLC 379 VFIAQGYTL NSCLC 381 KYPASSSVF RCC, NSCLC

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues, and a high tumor-to-normal ratio of geneexpression indicates a therapeutic window. Moreover, over-expression ofsource genes in tumor entities that were not yet analyzed for peptidepresentation indicates that a certain peptide may be of importance inthe respective entity.

For HLA class I-binding peptides of this invention, normal tissueexpression of all source genes was shown to be minimal based on adatabase of RNASeq data covering around 3000 normal tissue samples(Lonsdale, 2013). In addition, gene expression data from tumors vsnormal tissues were analyzed to assess target coverage in various tumorentities.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA and Royston, Herts, UK); Bio-OptionsInc. (Brea, Calif., USA); ProteoGenex Inc. (Culver City, Calif., USA);Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori“Pascale” (Naples, Italy); University Hospital of Heidelberg(Heidelberg, Germany); BioCat GmbH (Heidelberg, Germany), BioServe(Beltsville, Md., USA), Capital BioScience Inc. (Rockville, Md., USA).

Total RNA from tumor tissues for RNASeq experiments was obtained from:Asterand (Detroit, Mich., USA & Royston, Herts, UK), Bio-Options Inc.(Brea, Calif., USA), BioServe (Beltsville, Md., USA), Geneticist Inc.(Glendale, Calif., USA), ProteoGenex Inc. (Culver City, Calif., USA),Tissue Solutions Ltd (Glasgow, UK), University Hospital Bonn (Bonn,Germany), University Hospital Heidelberg (Heidelberg, Germany),University Hospital Tübingen (Tübingen, Germany)

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (Illumina Inc,San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in differentcancers are shown in FIGS. 2A-2D. Expression scores for furtherexemplary targets are shown in Table 10 (A and B), based on in-houseRNASeq analyses. Expression data for other entities and furtherexemplary peptides are summarized in Table 11, based on data generatedby the TCGA Research Network: cancergenome.nih.gov/.

TABLE 10ATarget coverage within various tumor entities, for expression of source genes of selected peptides. Over-expression was defined as more than 1.5-fold higher expression on a tumor compared to the relevant normal tissue that showed highest expression of the gene.<19% over-expression = I, 20-49% = II, 50-69% = III, >70% = IV. If a peptide could be derived from several source genes, the gene withminimal coverage was decisive. The baseline included the following relevant normal tissues: adipose tissue, adrenal gland, artery, bonemarrow, brain, cartilage, colon, esophagus, gallbladder, heart, kidney, liver, lung, lymph node, pancreas, pituitary, rectum, skeletalmuscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea, urinary bladder and vein. In case expression data forseveral samples of the same tissue type were available, the arithmetic mean of all respectivesamples was used for the calculation. SEQ ID AML BRCA CLL CRC GB HCCpNSCLC OC OSCAR PC RCC SCLC NO Sequence (N = 7) (N = 10) (N = 10)(N = 20) (N = 24) (N = 15) (N = 11) (N = 12) (N = 11) (N = 26) (N = 10)(N = 10) 2 ALYGKLLKL I II I I I I I I I I I I 3 TLLGKQVTL I II I I I I II I I I I 5 SLFGQEVYC I I I I I II I I I I I I 9 FLGDYVENL I I IV I IIII I I I I I II 10 KTLDVFNIIL I I IV I III I I I I I I II 11 GVLKVFLENV III I I I I I II I I I I 12 GLIYEETRGV I II I I I I I II I I I I 13VLRDNIQGI I II I I I I I II I I I I 14 LLDHLSFINKI I I III I I I I I I II III 16 HLYNNEEQV I I I I I IV I I II I II I 17 ILHEHHIFL I I I II I IIII II I II IV I 18 YVLNEEDLQKV I I I II I II II II I II IV I 19TLLPTVLTL I I I I I I I I I I IV I 20 ALDGHLYAI I I I I I I I I I I IV I27 KVFGDLDQV I I I I I I I I I I II I 28 TLYSMDLMKV I I I I I I I I I III I 31 ALSDETKNNWEV I III I II III I III III II I I IV 32 ILADEAFFSV IIII I II III I III III II I I IV 33 LLLPLLPPLSPSLG I I I I I I I I I I III 36 KLYGIEIEV I I I I II I I I I I I I 39 ALMAVVSGL I I IV I I I I I II I I 42 VLSPFILTL I I I I I I I I II I I I 44 GLLWQIIKV I I I III I I II I I I I 45 VLGPTPELV I I I I I I I I I II I I 46 SLAKHGIVAL I II I I II I I I I I I 47 GLYQAQVNL I I I II II II III I IV II I II 49 LLDESKLTLI I I I I I I I I I III I 50 EYALLYHTL I I I II I I I I I I II I 51LLLDGDFTL I I I I I III I I I I I I 52 ELLSSIFFL I I I I I I II I II III II 53 SLLSHVIVA I II I II I I II II II I I II 54 FINPKGNWLL I I I I II II I I II I I 55 IASAIVNEL I II II I II I II II I I I II 59 ALTKILAELI I I I I I I I I I I II 63 TLSSERDFAL I I I I I III I I I I II I 64GLSSSSYEL I I I I I III I I I I I I 65 KLDGICWQV I I I I I II I I I I II 67 GVIETVTSL IV I I I I I I I I I I I 69 GIYDGILHSI I I I II II II IIII II I I II 70 GLFSQHFNL III I I I I I I I II I I I 73 GVPDTIATL I I III I I I I I I I I 75 ILDNVKNLL IV I I I I I I I I I I I 78 LLWGHPRVA IIV I II I I III III IV III I I 79 SLVPLQILL I I I I I II I I I I I I 80TLDEYLTYL I I I I I II I II I I I I 81 VLFLGKLLV I I II II III II I IIII II II IV 84 FLEEEITRV I I I I I I I I I I I II 86 LLVTSLVW I I I I IIII I I I I I I 88 ILLNTEDLASL I I I I I I I I I I II II 91 SSLEPQIQPV III II I II I I II III I II I 322 ALVSGGVAQA I III I I I I I III III II II 323 ILSVVNSQL I I IV II I I I I I I I I 324 AIFDFCPSV I I IV I I I I II I I I 325 RLLPKVQEV I I I I II I I I I I I I 327 SIGDIFLKY I II I IIIII I IV III III II I IV 328 SVDSAPAAV I I I I I I I I I I I II 329FAWEPSFRDQV I I I I I III I I I I I I 331 AIWKELISL I I I I II I I I I II III 332 AVTKYTSAK I II I I I II I I I I I II 334 GRADALRVL I I IV I II I I I I I I 335 VLLAAGPSAA I I II I I I I I I I I II 336 GLMDGSPHFL III I I I I I I I I I I 337 KVLGKIEKV I II I I I I I I I I I II 338LLYDGKLSSA I II I I I I I I I I I II 96 YYTQYSQTI I IV I II I I III IIIIV III I I 98 VFPRLHNVLF I I I I I I I I I I I II 100 VYIESRIGTSTSF I III I I I I II II I I II 101 IYIPVLPPHL I IV I I I I IV III III I I I 103NYIPVKNGKQF I I III I I I I I I I I I 106 IYNETVRDLL I I I I I I I II II I I 107 KYFPYLWI I II I I I I I II III I II I 108 PYLWIHTL I II I I II I II III I II I 110 SYPKIIEEF I I III I I I I I I I I I 113 IYSFHTLSFI I I I II I I I I I I III 114 QYLDGTWSL I IV I II II I IV II IV III III 115 RYLNKSFVL I II I II I I I III I I I II 117 IYLSDLTYI I I IV I I II I I I I I 118 KYLNSVQYI I I IV I I I I I I I I I 119 VYRVYVTTF I I I II I II I II I I I 121 RYGLPAAWSTF I I IV I I I I I I I I I 123 VYTPVLEHLI III I II III I III III II I I IV 124 TYKDYVDLF I III I II III I IIIIII II I I IV 125 VFSRDFGLLVF I III I II III I III III II I I IV 126PYDPALGSPSRLF I I I II I III I II I I I I 127 QYFTGNPLF I I I I I I I II I I II 132 RYPALFPVL I I I I I I I II I I I I 135 VYEAMVPLF I I I I II I I I I I II 138 VYNAVSTSF I I I I I I I I I I I II 139 IFGIFPNQF IV II I I I I I I I II I 140 RYLINSYDF I I I I I I II I I I I II 141SYNGHLTIWF I I I I III I I I I I I I 146 NYYERIHAL III I IV I II I II III I I I 147 LYLAFPLAF I II I I I I I I I I I I 151 AYISGLDVF I I I I I III I IV I I II 152 KFFDDLGDELLF I I I I I I II I IV I I II 156YYMELTKLLL I I I I I I I I III I I IV 157 DYIPASGFALF I I I I II I I I II I I 158 IYEETRGVLKVF I II I I I I I II I I I I 159 IYEETRGVL I II I II I I II I I I I 162 KYTSYILAF I I I I I I I II I I II I 164 HYVPATKVF III I I I I IV IV III I II II 167 RYGFYYVEF I I I I IV I III II II I I I171 NYEDHFPLL I I I I I I I II II I I II 172 VFIFKGNEF I I I I I I II III I I I 173 QYLEKYYNL I I I I I I II I II I I I 174 VYEKNGYIYF I II I II I II I III I I I 175 LYSPVPFTL I I I I I I I I II I I I 176 FYINGQYQFIII I III I II I II I II I I II 177 VYFKAGLDVF I II I II I I I II I I II 178 NYSSAVQKF I I I I III I I I I I I I 179 TYIPVGLGRLL I I I I I I IIII II I I II 181 VYPPYLNYL I II I I I I I I I I I I 182 AYAQLGYLLF I II I I I I I I I I II 184 IYSVGAFENF I II I I I I I I I I I II 185QYLVHVNDL I I I I II I I I I I II II 189 AYFKQSSVF II I II I I I I I I II I 190 LYSELTETL IV III IV I II I IV I I II I IV 191 TYPDGTYTGRIF I III I I I I I I I I I 193 LYLENNAQTQF I I I I I I II I IV I I I 195AYIKGGWIL I I I I I II I I I I I I 198 DYVGFTLKI I I I I III I II I I II I 203 VYNVEVKNAEF I I I I I I I I I I I II 204 EYLSTCSKL II II I I I II II I I I III 205 VYPWLNQI I I I I I I I I I I I II 206 NYLDVATFL I I II II I II I I I I II 208 TYLEKIDGF I I I I I I I I II I I I 210IYAGVGEFSF I I I I I I I I I I I II 211 VFKSEGAYF I I I I I I I I I I III 212 SYAPPSEDLF I II I I I I I I I I I I 213 SYAPPSEDLFL I II I I I II I I I I I 214 KYLMELTLI I I I I I I I I I I I II 216 FYVNVKEQF I I I II I I I I I I II 218 LYSELNKWSF I I I I I I II I I I I I 219 SYLKAVFNL IIII I I I I I I I I I I 220 SYSEIKDFL I I IV I I I I I I I I I 221KYIGNLDLL I I I I I I I I I I II I 222 HYSTLVHMF I I I I I I I I I I III 224 PYFFANQEF II I I I I I I I I I I I 226 MYLKLVQLF I I I I I I I I II I II 227 IYRFITERF I I I I I I I I I I I II 230 TYGMVMVTF I I I I II II I I I I I 231 AFADVSVKF I I I I II I I I I I I I 233 QYLTAAALHNL I I II I I I I I I I I 235 VYKDSIYYI II I IV I I I I I I I I I 236 VYLPKIPSWI I I I I II I I I I I I 237 KYVGQLAVL I I I I I I I I I I I II 239VYAIFRILL I II I I I I I I I I I II 240 YYFFVQEKI I I I I II I I I I I II 241 SYVKVLHHL I I I I I I I I I I I II 242 VYGEPRELL I II I I I II I II I I II 243 SYLELANTL I I I I I I I I II I I I 244 VHFEDTGKTLLF I II II I I II I III I I I 245 LYPQLFWL I I I I I I I II I I I I 246 KYLSVQLTLI I I I I I I I I I I II 247 SFTKTSPNF I I I II I I I I I I I I 251VYTKALSSL I I I I I I I I I I I III 256 VFGKSAYLF II I I I I I I I I I II 258 AYAQLGYLL I I I I I I I I I I I II 263 RYILENHDF I I I I I II I II I I I 267 SFLNIEKTEILF I I I I I III I I I I I I 270 YYSQESKVLYL I IIII I II I I III II I I II 272 RYVFPLPYL I I I II I II I I I I I I 273IYGEKLQFIF I I I I I I I I I I I II 274 KQLDIANYELF I III I I II I I III I I II 276 QYLDVLHAL I II I I I I I II II I II II 277 FYTFPFQQL I I II I I I I I I I II 278 KYVNLVMYF I I I I I I I II I I I I 279 VWLPASVLFI I I I I I I I I I I II 282 NYLVDPVTI I II I I I I I III I I I I 283EYQEIFQQL I II I I I I I III I I I I 287 IYNPNLLTASKF I III I I I I IIII III I I I 291 YYLGSGRETF I I I I II I II II III I I II 292 FYPQIINTFI I I I II I I I I I I I 293 VYPHFSTTNLI I I I I II I I I I I I I 294RFPVQGTVTF I IV I I I I I II I I I I 295 SYLVIHERI I I II I II I I III II I II 300 TYPSQLPSL I I I II I I I I I I I I 302 LYLPVHYGF I I I II I II I I I I II 304 EYNEVANLF I I I I II I I I I I I I 307 GYAFTLPLF I II III II I I II I I II II 308 TFDGHGVFF I I I II I I I I II I I I 309KYYRQTLLF I I I I I I I III IV I II II 310 IYAPTLLVF I II II I I I I I II I I 314 RYFKGDYSI I I I I II I I I I I I I 315 FYIPHVPVSF I I I I IIII I I I I I I 320 KFILALKVLF I I I I I I I I I I I II 342 NYAFLHRTL III I I I I I II III I I I 343 NYLGGTSTI I II I I I II I I I I I II 344EYNSDLHQF I I I I I I I I I I I II 345 EYNSDLHQFF I I I I I I I I I I III 348 IYLEHTESI I I II I I I I I I I I I 350 KYGNFIDKL I I I I I I I III I I I 351 IFHEVPLKF I I I I I I I I I I I II 352 QYGGDLTNTF I I I I II II I I II I I 353 TYGKIDLGF I I I I I I I I I I I II 354 VYNEQIRDLL II I I I I I I I I I II 355 IYVTGGHLF I I I I I I I I II I I I 356NYMPGQLTI I I I II I I II II I II II I 358 YYSEVPVKL I IV I II I I IIIIII IV III I I 359 NYGVLHVTF II I II II II III I I I I II II 360VFSPDGHLF I II I I I I I I I I I I 361 TYADIGGLDNQI II I I I I I I I I II I 363 SYAELGTTI I I I I II I I I II I I I 364 KYLNENQLSQL I I I I I II I I I I III 365 VFIDHPVHL I I I I II I I I I I I II 366 QYLELAHSL I II I I I I II I I I II 367 LYQDHMQYI I I I I I I I I II I I I 368KYQNVKHNL I I I I I I I I I I I II 371 AYSHLRYVF I I I IV III I IV II IVIII III II 372 VYVIEPHSMEF I I II I I I I I I I I II 374 VFLPRVTEL I IIII II III I III III II I I IV 376 VYTPVASRQSL I I I I II I I I I I I II377 QYTPHSHQF I I I I II I II I I I I III 378 VYIAELEKI I II I I I I I II I I II 379 VFIAQGYTL I I I I I I II I II I II II 380 VYTGIDHHW I II III I I II III IV I I III 381 KYPASSSVF I I I I I I I II I I III I 382AYLPPLQQVF I I I I I I I I I I I II 383 RYKPGEPITF II IV I II I I IV IIII I I II 385 QYIEELQKF I I IV I I I I I I I I I 386 TFSDVEAHF I I I I II I I I I I II 387 KYTEKLEEI I I I I IV I I II I I I III

TABLE 10BTarget coverage for source genes of selected peptides. Over-expression wasdefined as more than 1.5-fold higher expression on a tumor compared to the relevant normaltissue that showed highest expression of the gene. <19% over-expression = I, 20-49% = II,50-69% = III, >70% = IV. If a peptide could be derived from several source genes, the genewith minimal coverage was decisive. The baseline included the following relevant normaltissues: adipose tissue, adrenal gland, artery, bone marrow, brain, cartilage, colon,esophagus, gallbladder, heart, kidney, liver, lung, lymph node, pancreas, pituitary, rectum,skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea,urinary bladder and vein. In case expression data for several samples of the same tissuetype were available, the arithmetic mean of all respective samples was used for thecalculation. NHL = non-Hodgkin lymphoma, PCA = prostate cancer and benign prostatehyperplasia, GC = gastric cancer, GBC_CCC = gallbladder adenocarcinoma andcholangiocarcinoma, MEL = melanoma, UBC = urinary bladder cancer, UTC = uterinecancer, HNSCC = head and neck small cell carcinoma. SEQ GBC_ ID NHL OCAGC CCC MEL UBC UTC HNSCC NO. Sequence (N = 10) (N = 10) (N = 11)(N = 10) (N = 10) (N = 10) (N = 10) (N = 10) 2 ALYGKLLKL I I I I I I I I3 TLLGKQVTL I I I I I I I I 4 ELAEIVFKV I I I I I I I II 9 FLGDYVENL I II I I I I I 12 GLIYEETRGV II II I I II I I I 13 VLRDNIQGI II II I I II II I 19 TLLPTVLTL I I I I I III I I 32 ILADEAFFSV II I I I II I I I 34LLPKKTESHHKT II I I I I I I I 35 YVLPKLYVKL I I I I I I I I 37ALINDILGELVKL I I I I I I I I 39 ALMAVVSGL II I I I I I I I 41 FVLPLVVTLI I I I I I I I 42 VLSPFILTL I I I II I II I II 45 VLGPTPELV I I II II II I I 46 SLAKHGIVAL I I I I I I II I 47 GLYQAQVNL I I I II IV II I III51 LLLDGDFTL I I I I I I I I 53 SLLSHVIVA III I I II I I II I 55IASAIVNEL II I I II I I I I 60 FLIDTSASM I I I I I I I I 61 HLPDFVKQL II I I I I I I 70 GLFSQHFNL I I I II I II III II 96 YYTQYSQTI I I I II II I II 98 VFPRLHNVLF I I I I I I I I 99 QYILAVPVL I I I I I I I I 100VYIESRIGTSTSF I I I I II I I I 101 IYIPVLPPHL III III I III I II III III103 NYIPVKNGKQF I I I I I I I I 104 SYLTWHQQI I I I I I I I I 105IYNETITDLL I I I I I I I I 110 SYPKIIEEF II I I I I I I I 113 IYSFHTLSFI I I I I I I II 114 QYLDGTWSL II I II IV IV III IV III 116 AYVIAVHLF II I I I I I I 117 IYLSDLTYI II I I I I I I I 118 KYLNSVQYI II I I I I II I 119 VYRVYVTTF I I I I I I II I 121 RYGLPAAWSTF I I I I I I I I 123VYTPVLEHL II I I I II I I I 124 TYKDYVDLF II I I I II I I I 126PYDPALGSPSRLF I II II II I I III I 127 QYFTGNPLF I I I I I I I I 128VYPFDWQYI I I I I I I I I 132 RYPALFPVL I I I I I I I I 135 VYEAMVPLF II I I I I I I 144 VFASLPGFLF III I I I I I I I 145 VYALKVRTI II I I IIII II I II 147 LYLAFPLAF I I I I I I I I 150 IYITRQFVQF I I I I II I I I151 AYISGLDVF I I I I I I I III 155 IYKWITDNF I I I I I I II I 156YYMELTKLLL II I I II I I I I 158 IYEETRGVLKVF II II I I II I I I 161KYPDIVQQF II I I I I I I I 162 KYTSYILAF II I I II I I I I 163RYLTISNLQF I I I I I I I I 165 EYFTPLLSGQF I I I I I I I I 166 FYTLPFHLII I I I I I I I 168 RYLEAALRL I I I I II I I I 170 QYPFHVPLL III I I I II I I 171 NYEDHFPLL I I II I IV II I II 172 VFIFKGNEF I I I I I I I I175 LYSPVPFTL I I I II I I I II 176 FYINGQYQF I II I I I I I II 177VYFKAGLDVF I III I II I II II I 178 NYSSAVQKF I III I I II I I I 179TYIPVGLGRLL I I I I I I III II 180 KYLQVVGMF I I I I I I I I 191TYPDGTYTGRIF II I I I I I I I 195 AYIKGGWIL I I I I I I I I 197IFTDIFHYL I I I I I I I I 204 EYLSTCSKL II II I I I I IV II 206NYLDVATFL I I I I I I II I 235 VYKDSIYYI IV I I I I I I I 277 FYTFPFQQLI I I I I I I II 291 YYLGSGRETF I I I II II II III III 296 SYQVIFQHF I II I I I I II 297 TYIDTRTVF I I I I I I III I 304 EYNEVANLF I III I I I II I 307 GYAFTLPLF I II I I I I II I 309 KYYRQTLLF I I I I I I II I 312SYTSVLSRL II I I I II I I I 316 VYFEGSDFKF II I I I I I I I 317VFDTSIAQLF I I I I I I I I 318 TYSNSAFQYF I I I I I I I I 319 KYSDVKNLII I I I I I I I 326 SLLPLVWKI I I I I I I I I 327 SIGDIFLKY I I II III II III II 328 SVDSAPAAV II I I I I I I I 330 FLWPKEVEL II I I I I I I I331 AIWKELISL I I I I I I I II 332 AVTKYTSAK I I I I I I I I 333GTFLEGVAK I I I I I I I I 334 GRADALRVL I I I I I I I I 335 VLLAAGPSAAIII I I I I I I I 342 NYAFLHRTL I I I I I II I I 343 NYLGGTSTI I IV I II I II I 344 EYNSDLHQF II I I I I I I I 345 EYNSDLHQFF II I I I I I I I347 VYAEVNSL I I I I I I I I 348 IYLEHTESI I I I I I I I I 350 KYGNFIDKLI I I I I I I I 352 QYGGDLTNTF I I I II I I I 353 TYGKIDLGF II I I I I II I 354 VYNEQIRDLL I I I I I I I I 355 IYVTGGHLF I I I II I II I II 356NYMPGQLTI I I II II I I I I 359 NYGVLHVTF IV I I I II I I I 360VFSPDGHLF II I I I I I I I 361 TYADIGGLDNQI I I I I I I I I 362VYNYAEQTL I I I I I I I I 363 SYAELGTTI I I I I I I I I 365 VFIDHPVHL II I I I I I I 366 QYLELAHSL I I I I I I I I 367 LYQDHMQYI I I I I I I II 371 AYSHLRYVF I I III II I IV I II 376 VYTPVASRQSL II I I II I II I II380 VYTGIDHHW II I I II I I I II 383 RYKPGEPITF II III I III I II III II386 TFSDVEAHF II I I I I I I I 387 KYTEKLEEI I I I I I I I I

TABLE 8 Target coverage within various tumor entities, for expression ofsource genes of selected peptides. A gene was considered over-expressedif its expression level in a tumor sample was more than 2-fold above thehighest 75% percentile of expression levels determined from samples ofthe following normal organs (adjacent to tumors): rectum (n = 10),esophagus (n = 11), bladder (n = 19), kidney (n = 129), stomach (n =35), colon (n = 41), head and neck (n = 43), liver (n = 50), lung (n =51), thyroid (n = 59), lung (n = 59). Over-expression categories areindicated as “A” (>=50% of tumors above the cutoff), “B” (>=20% oftumors above the cutoff, but <50%), and “C” (> = 5% of tumors above thecutoff, but <20%). SEQ ACC BLCA CESC CHOL DLBC HNSC KICH KIRP LGG MESOPCPG PRAD SARC SKCM STAD TGCT THCA THYM UCEC UCS UVM ID (N = (N = (N =(N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N= (N = (N = (N = NO. 79) 408) 307) 36) 48) 521) 66) 291) 534) 87) 184)498) 263) 473) 415) 156) 513) 120) 546) 57) 80) 16 C C C C C 2 3 4 B C BB C C A C C 5 6 8 C C C 9 10 11 C B B B A C C C C B A C B B B C A A B C12 C B B B A C C C C B A C B B B C A A B C 13 C B B B A C C C C B A C BB B C A A B C 1 C B B C C C B A B C B 17 B A C C C A B 18 B A C C C A B21 C B 22 C B 24 C C C 27 C 28 C 29 C 30 C 31 C C B C C B B B C 32 C C BC C B B B C 34 C C B C B B B C 35 B 36 C C B C C C 37 38 C 39 C B C 40 CC A C C C C A A C B A 42 A A B B B C C C C C C A C 45 B 47 C C C C B C CB C C C 48 C C C 49 51 C C C 52 C 53 54 C C C C C C C 55 C 56 C 58 C B C60 C C B C C C C 61 62 B C C C C 63 64 C 65 66 C B A C C C C C 67 B C B68 C 69 C 70 B B C B B C C C A B A A 72 C C B C B C C B C C C 74 A A A75 B 76 C 78 C C C B B B C C C 79 C 80 81 C C C C C C C C C C 84 A 86 87C C 88 C A B 90 C C B A C A 91 C C C C 92 B C C C C C C 321 B B C C B BC A B B C B B C B B B B 323 B 325 C B C B A C B C B C 326 A C C C 327 BA C A C B C A A B B 328 C B C C C B A C C B 329 330 C C C A C C A C C AA C C 331 B 332 C B A B B B C B A B B B A C B A B B 334 A C C C C 335 BA C A C C C C B B C A B C 336 340 B B C 175 C C B C C 97 C B 98 C C B CB 100 C B A C B B B B B A B B A 101 103 104 105 B A B B B C A B B C B106 B A A B B B C A A B A 107 C B 108 C B 110 B A A C C C B C A B B B111 C A B B C C C A B C 112 C A B B C C C A B C 113 B 114 C B C C C C BB B C C C C C 115 C 116 B B A B C B A B C C 117 C B C 118 C B C 119 C121 A C C C C 122 123 C C B C C B B B C 124 C C B C C B B B C 125 C C BC C B B B C 126 C C C B C B B B 127 C 128 C B C 129 B B C 130 B B C 131B 132 C C 133 C 134 B C B C C B A C B B 135 C C C C 136 C 137 138 B A CB B C C C A A B C B 139 B C B 140 141 142 C B B B C B C A A C C 144 C C145 C C C C B 146 B C 147 C 148 C 149 C 150 C A 151 C A B 152 C A B 153155 C B C 156 C C A A B C C C B B C C 157 B B C C 158 C B B B A C C C CB A C B B B C A A B C 159 C B B B A C C C C B A C B B B C A A B C 161 CB 162 C B A C C C B C B C C 164 B B C A B B B B B B A C C A B 165 C 166C 167 C C B 168 C C B C C C A 169 170 C C A C C C C C B B 171 B C C B CC C C 172 C B A C C C 173 C B A C C C 174 B B C A C B B C C C C C 96 C CC B B B C C C 176 177 B 178 A C C 180 C A C B A C C C C C C C 181 C B CA C 182 C 183 C C 184 185 C C C C C 186 C C C 187 C C C A C C C C C C C188 C B B B B B B 189 190 191 A C C C 192 B C B C A C B C 193 C B 194 BA C A B C B B B A A B B 195 196 197 C 198 C 199 C B B C C 200 A 202 C203 C C A C C C C C B C A C C B 204 205 C C 206 C C 207 C C C C 208 C CB C A C 209 B C 210 B A B B C C B B B A B B B 211 B B A B C C C A B B AB B A 212 C C 213 C C 214 B A B B C B C B A A C B 216 C B A B B C B B CB B C B 217 C 218 C C 219 C C C C 220 C B 221 B C C 222 B C C 223 C 224B 225 C C C C C 226 C B A C A B B B A A C B B 227 C C C 228 A 229 C A230 B B C C 231 B B C C 232 C B A C A B C C B A B A A B A 233 B C B 234C C C C C 235 A C 236 237 C A A C A A C B B A A A A B A 238 C C B C C B240 C C C B 241 C B C C C A B C C C 242 C C C C C B C B C 243 C C 244 BB C A C B B C C C C C 247 C C C A 249 C B 250 C A C C B B C C B 251 C BA C A B C C A B B A A B A 253 C B B C C C C C 255 C B 256 C B C 258 C259 C C C B B 262 C B C B 263 B 264 C A C C C B C 265 C 266 C B 267 C CC 268 269 C A A C B C B B C B C C 270 C C 271 C A 272 B C C C 273 C C BA C C C C B A A C B 274 C C C 276 A C C A 277 C C C C 278 B C C C 279 C282 C C C 283 C C C 284 C 285 C 286 B C C B 287 288 C C B B C C B C B AA C B 289 C C 291 C C C C C B 292 C C C B 293 C C C B 294 C C C 295 B C296 C C C C 297 C C C B C C C 298 C 300 A A B A B 301 C C 302 B 304 305C B C A B C 306 C C C B B C C C B C C 307 C C C C C C 309 B C C C 312 C313 C C 314 B 315 C 316 C 317 B C 319 B 320 C B B B B B B B A C C A 341B C C 342 C C C C C 343 C B 344 B B A B C B B A A B B A 345 B B A B C BB A A B B A 346 C B B B C B C A A C C 347 C 348 B A A C C C B C A B B B349 C B 350 C C 351 352 C C C C C C C 353 C B A C A A C B B B A A B B A354 C B A C B B B B B A A B B A 355 A A B B B C C C C C C A C 356 C B C357 C B C C C B C A C 358 C C C B B B C C C 359 C B C C 360 C C B C C CB 361 C C C C C B C B C 363 C C C C B C 364 B A C C B B C A A C B C 365C 366 C B A C B B C B A A C B A C 367 368 C 369 C B A B B B C B C B B B371 C B C C 372 B A C A C C C C B B C A B C 373 C 374 C C B C C B B B C375 B C 376 C C C C C C B C B 377 C C 378 C A C C C C C C 379 C 380 C CC C C C B C B C 382 C 383 385 C B C 386 C C C C B B C C 387 B B C C C388 C 389 C B B C C 390 C B A C B B A B B A A B C B 391 C B A B B B C BC B B B 392 B B A C B B C A B B B A B C B 393 C A 394 B A C A B C C B BB A A B B 395 C C B 396 A B C C C 397 A A C A A C C B B B A A B A 398 AA C A A C C B B B A A B A 399 B A C C C A B 400 B B B A C C C A C C 401B B B A C C C A C C 402 C A A A C A C A A C C A B A B B B A A 403 C B BB B B 404 C B B B B B 405 C B B B B B 406 C B C C C B C A C 407 C A A CA B C C B B B A A B A 408 B A B B B C B A B B B 409 B A B B C C B B B C410 C A A C A B C C B B B A A B A 412 C B A C B B A B B A A B C B 413 C414 A C C 415 C C B C C C C B B C C C C 416 C 417 C B A B B C B B A A BB ACC = Adrenocortical carcinoma (N = 79), BLCA = Bladder urothelialcarcinoma (N = 408), LGG = Lower grade glioma (N = 534), CESC = Cervicalsquamous cell carcinoma and endocervical adenocarcinoma (N = 307), STAD= Stomach adenocarcinoma (N = 415), CHOL = Cholangiocarcinoma (N = 36),MESO = Mesothelioma (N = 87), KICH = Kidney chromophobe (N = 66), PRAD =Prostate adenocarcinoma (N = 498), DLBC = Lymphoid neoplasm diffuselarge B-cell lymphoma (N = 48), PCPG = Pheochromocytoma andparaganglioma (N = 184), KIRP = Kidney renal papillary cell carcinoma (N= 291), SKCM = Skin cutaneous melanoma (N = 473), SARC = Sarcoma (N =263), THCA = Thyroid carcinoma (N = 513), THYM = Thymoma (N = 120), UCS= Uterine carcinosarcoma (N = 57), UCEC = Uterine corpus endometrialcarcinoma (N = 546), UVM = Uveal melanoma (N = 80), TGCT = Testiculargerm cell tumors (N = 156), HNSC = Head and neck squamous cell carcinoma(N = 521)

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*02:01 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 9).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 418) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.419), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Different Cancer Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIGS. 3A-3B together withcorresponding negative controls. Results for 5 peptides from theinvention are summarized in Table 12A. Exemplary flow cytometry resultsafter TUMAP-specific multimer staining for 7 peptides of the inventionare shown in FIGS. 4A-4C and FIGS. 5A-5D together with correspondingnegative controls. Results for 74 peptides from the invention aresummarized in Table 12B.

TABLE 9A in vitro immunogenicity of HLA class I peptidesof the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20 % = +; 20%-49% = ++;50%-69 % = +++; > = 70% = ++++ Seq ID Sequence wells 393 KYIEAIQWI ++399 SYIDVLPEF ++ 400 KYLEKYYNL ++ 407 VYGIRLEHF +++ 414 MYPYIYHVL ++

TABLE 12B in vitro immunogenicity of HLA class I peptidesof the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +; 20%-49% = ++;50%-69% = +++; > = 70% = ++++ Seq ID Sequence Wells positive [%]   2ALYGKLLKL ++++   7 AAAAKVPEV +   8 KLGPFLLNA +++   9 FLGDYVENL +  17ILHEHHIFL +  43 LLWAGPVTA ++++ 322 ALVSGGVAQA + 331 AIWKELISL ++  96YYTQYSQTI +  98 VFPRLHNVLF +  99 QYILAVPVL +++ 102 VYPFENFEF +++ 103NYIPVKNGKQF + 104 SYLTWHQQI + 105 IYNETITDLL + 106 IYNETVRDLL + 107KYFPYLVVI ++ 109 LFITGGQFF ++ 110 SYPKIIEEF ++ 111 VYVQILQKL + 112IYNFVESKL +++ 114 QYLDGTWSL +++ 115 RYLNKSFVL + 119 VYRVYVTTF +++ 120GYIEHFSLW ++ 122 EYQARIPEF ++ 132 RYPALFPVL + 137 EYLHNCSYF + 139IFGIFPNQF ++ 140 RYLINSYDF +++ 142 VYVDDIYVI ++++ 144 VFASLPGFLF ++ 155IYKWITDNF ++ 156 YYMELTKLLL + 157 DYIPASGFALF + 158 IYEETRGVLKVF + 160RYGDGGSSF + 161 KYPDIVQQF + 162 KYTSYILAF + 163 RYLTISNLQF + 164HYVPATKVF + 166 FYTLPFHLI ++++ 167 RYGFYYVEF ++++ 168 RYLEAALRL +++ 170QYPFHVPLL +++ 171 NYEDHFPLL ++ 172 VFIFKGNEF + 174 VYEKNGYIYF ++++ 175LYSPVPFTL + 177 VYFKAGLDVF + 179 TYIPVGLGRLL +++ 180 KYLQVVGMF + 181VYPPYLNYL ++++ 182 AYAQLGYLLF +++ 186 VFTTSSNIF + 190 LYSELTETL ++++ 277FYTFPFQQL +++ 344 EYNSDLHQF + 345 EYNSDLHQFF ++ 349 QYSIISNVF ++ 350KYGNFIDKL +++ 351 IFHEVPLKF ++ 353 TYGKIDLGF + 354 VYNEQIRDLL + 356NYMPGQLTI + 358 YYSEVPVKL ++++ 359 NYGVLHVTF + 360 VFSPDGHLF ++ 363SYAELGTTI + 365 VFIDHPVHL + 366 QYLELAHSL ++ 367 LYQDHMQYI ++ 371AYSHLRYVF ++ 380 VYTGIDHHW +

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1h at37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 13 MHC class I binding scores. Binding of HLA-classI restricted peptides to HLA-A*02 was ranged bypeptide exchange yield: <20% = +; 20%-49% = ++;50%-75% = +++; > = 75% = ++++ Seq ID Sequence Peptide exchange 1PLWGKVFYL ++ 2 ALYGKLLKL +++ 3 TLLGKQVTL +++ 4 ELAEIVFKV +++ 5 SLFGQEVYC+++ 6 FLDPAQRDL +++ 7 AAAAKVPEV +++ 8 KLGPFLLNA +++ 9 FLGDYVENL ++ 10KTLDVFNIIL ++ 11 GVLKVFLENV ++ 12 GLIYEETRGV ++ 13 VLRDNIQGI +++ 14LLDHLSFINKI ++ 16 HLYNNEEQV ++ 17 ILHEHHIFL +++ 18 YVLNEEDLQKV +++ 19TLLPTVLTL +++ 20 ALDGHLYAI +++ 21 SLYHRVLLY ++++ 22 MLSDLTLQL ++++ 23AQTVVVIKA + 24 FLWNGEDSAL +++ 25 IQADDFRTL ++ 26 KVDGVVIQL +++ 27KVFGDLDQV +++ 28 TLYSMDLMKV +++ 29 TLCNKTFTA +++ 31 ALSDETKNNWEV ++++ 32ILADEAFFSV +++ 33 LLLPLLPPLSPSLG +++ 35 YVLPKLYVKL ++ 36 KLYGIEIEV ++++37 ALINDILGELVKL +++ 38 KMQEDLVTL +++ 39 ALMAVVSGL +++ 40 SLLALPQDLQA+++ 41 FVLPLVVTL +++ 42 VLSPFILTL +++ 43 LLWAGPVTA +++ 44 GLLWQIIKV ++45 VLGPTPELV +++ 46 SLAKHGIVAL +++ 47 GLYQAQVNL +++ 48 TLDHKPVTV ++ 49LLDESKLTL +++ 50 EYALLYHTL ++ 51 LLLDGDFTL +++ 52 ELLSSIFFL +++ 53SLLSHVIVA +++ 54 FINPKGNWLL +++ 55 IASAIVNEL ++ 56 KILDLTRVL ++ 57VLISSTVRL ++ 58 ALDDSLTSL ++ 59 ALTKILAEL +++ 60 FLIDTSASM ++ 61HLPDFVKQL ++ 62 SLFNQEVQI +++ 63 TLSSERDFAL + 64 GLSSSSYEL ++ 65KLDGICWQV +++ 66 FITDFYTTV +++ 67 GVIETVTSL ++ 69 GIYDGILHSI +++ 70GLFSQHFNL +++ 71 GLITVDIAL +++ 72 GMIGFQVLL +++ 74 ILDETLENV ++ 75ILDNVKNLL +++ 76 ILLDESNFNHFL +++ 77 IVLSTIASV +++ 78 LLWGHPRVA +++ 79SLVPLQILL ++++ 80 TLDEYLTYL +++ 81 VLFLGKLLV ++ 82 VLLRVLIL ++ 83ELLEYLPQL +++ 84 FLEEEITRV +++ 85 STLDGSLHAV +++ 87 YLTEVFLHVV +++ 88ILLNTEDLASL +++ 89 YLVAHNLLL +++ 90 GAVAEEVLSSI + 91 SSLEPQIQPV + 92LLRGPPVARA ++ 93 SLLTQPIFL +++ 321 SLWFKPEEL +++ 322 ALVSGGVAQA +++ 323ILSVVNSQL +++ 324 AIFDFCPSV ++++ 325 RLLPKVQEV ++ 326 SLLPLVWKI +++ 327SIGDIFLKY +++ 328 SVDSAPAAV ++ 329 FAWEPSFRDQV ++ 330 FLWPKEVEL +++ 331AIWKELISL +++ 333 GTFLEGVAK +++ 334 GRADALRVL +++ 335 VLLAAGPSAA ++ 336GLMDGSPHFL ++ 337 KVLGKIEKV +++ 338 LLYDGKLSSA ++ 339 VLGPGPPPL ++ 340SVAKTILKR ++

TABLE 14 MHC class I binding scores. Binding of HLA-classI restricted peptides to HLA-A*24 was ranged bypeptide exchange yield: <20% = +; 20%-49% = ++;50%-75% = +++; > = 75% = ++++ Seq ID Sequence Peptide exchange  96YYTQYSQTI ++++  97 TYTFLKETF ++++  98 VFPRLHNVLF +++  99 QYILAVPVL ++++100 VYIESRIGTSTSF +++ 102 VYPFENFEF +++ 103 NYIPVKNGKQF +++ 104SYLTWHQQI ++++ 105 IYNETITDLL +++ 106 IYNETVRDLL +++ 107 KYFPYLVVI +++108 PYLVVIHTL +++ 109 LFITGGQFF ++++ 110 SYPKIIEEF +++ 111 VYVQILQKL +++112 IYNFVESKL +++ 113 IYSFHTLSF +++ 114 QYLDGTWSL ++++ 115 RYLNKSFVL +++116 AYVIAVHLF ++++ 117 IYLSDLTYI +++ 118 KYLNSVQYI +++ 119 VYRVYVTTF +++120 GYIEHFSLW ++++ 121 RYGLPAAWSTF +++ 122 EYQARIPEF +++ 123 VYTPVLEHL++ 124 TYKDYVDLF + 125 VFSRDFGLLVF +++ 127 QYFTGNPLF +++ 128 VYPFDWQYI++++ 129 KYIDYLMTW ++++ 131 EYLDRIGQLFF +++ 132 RYPALFPVL ++++ 133KYLEDMKTYF +++ 134 AYIPTPIYF +++ 135 VYEAMVPLF ++++ 136 IYPEWPVVFF +++137 EYLHNCSYF ++++ 138 VYNAVSTSF ++ 139 IFGIFPNQF +++ 140 RYLINSYDF ++++141 SYNGHLTIWF +++ 142 VYVDDIYVI +++ 143 KYIFQLNEI +++ 144 VFASLPGFLF++++ 145 VYALKVRTI +++ 146 NYYERIHAL +++ 147 LYLAFPLAF +++ 148 SYGTVSQIF++++ 149 SYGTVSQI ++++ 152 KFFDDLGDELLF ++ 153 VYVPFGGKSMITF ++++ 154VYGVPTPHF ++++ 155 IYKWITDNF ++++ 156 YYMELTKLLL ++++ 157 DYIPASGFALF+++ 158 IYEETRGVLKVF +++ 159 IYEETRGVL +++ 160 RYGDGGSSF +++ 161KYPDIVQQF +++ 162 KYTSYILAF ++ 163 RYLTISNLQF ++++ 164 HYVPATKVF +++ 165EYFTPLLSGQF +++ 166 FYTLPFHLI ++++ 167 RYGFYYVEF +++ 168 RYLEAALRL +++169 NYITGKGDVF +++ 170 QYPFHVPLL ++++ 171 NYEDHFPLL +++ 172 VFIFKGNEF++++ 173 QYLEKYYNL ++++ 174 VYEKNGYIYF +++ 175 LYSPVPFTL +++ 176FYINGQYQF +++ 177 VYFKAGLDVF +++ 178 NYSSAVQKF +++ 179 TYIPVGLGRLL +++180 KYLQVVGMF +++ 181 VYPPYLNYL +++ 182 AYAQLGYLLF ++++ 183 PYLQDVPRI+++ 184 IYSVGAFENF ++++ 185 QYLVHVNDL ++++ 186 VFTTSSNIF ++++ 187AYAANVHYL ++++ 188 GYKTFFNEF +++ 190 LYSELTETL +++ 191 TYPDGTYTGRIF +++192 RYSTFSEIF +++ 193 LYLENNAQTQF +++ 194 VYQSLSNSL +++ 195 AYIKGGWIL+++ 196 GYIRGSWQF ++++ 197 IFTDIFHYL ++++ 198 DYVGFTLKI ++ 199 SYLNHLNNL+++ 200 VFIHHLPQF +++ 201 GYNPNRVFF +++ 202 RYVEGIVSL +++ 204 EYLSTCSKL+++ 205 VYPVVLNQI +++ 206 NYLDVATFL ++++ 207 LYSDAFKFIVF +++ 208TYLEKIDGF ++++ 209 AFIETPIPLF ++++ 210 IYAGVGEFSF ++++ 211 VFKSEGAYF++++ 212 SYAPPSEDLF ++ 213 SYAPPSEDLFL ++ 214 KYLMELTLI +++ 215SYVASFFLL ++ 216 FYVNVKEQF +++ 217 IYISNSIYF ++++ 218 LYSELNKWSF +++ 219SYLKAVFNL +++ 220 SYSEIKDFL ++++ 221 KYIGNLDLL ++++ 223 TFITQSPLL ++++224 PYFFANQEF +++ 225 TYTNTLERL +++ 226 MYLKLVQLF ++ 227 IYRFITERF +++228 IYQYVADNF +++ 229 IYQFVADSF +++ 230 TYGMVMVTF +++ 231 AFADVSVKF ++++232 YYLSDSPLL +++ 233 QYLTAAALHNL +++ 234 SYLPAIWLL +++ 235 VYKDSIYYI+++ 236 VYLPKIPSW +++ 237 KYVGQLAVL +++ 239 VYAIFRILL +++ 240 YYFFVQEKI+++ 241 SYVKVLHHL +++ 242 VYGEPRELL +++ 243 SYLELANTL +++ 244VHFEDTGKTLLF +++ 245 LYPQLFVVL +++ 246 KYLSVQLTL ++ 247 SFTKTSPNF +++248 AFPTFSVQL ++++ 249 RYHPTTCTI ++++ 250 KYPDIASPTF ++ 251 VYTKALSSL+++ 252 AFGQETNVPLNNF ++++ 253 IYGFFNENF +++ 254 KYLESSATF +++ 255VYQKIILKF +++ 256 VFGKSAYLF +++ 257 IFIDNSTQPLHF +++ 258 AYAQLGYLL +++259 YFIKSPPSQLF ++ 260 VYMNVMTRL ++++ 261 GYIKLINFI ++++ 262 VYSSQFETI++++ 263 RYILENHDF +++ 264 LYTETRLQF ++++ 265 SYLNEAFSF ++++ 266KYTDVVTEFL +++ 267 SFLNIEKTEILF ++ 268 IFITKALQI ++ 269 QYPYLQAFF +++270 YYSQESKVLYL +++ 271 RFLMKSYSF ++++ 272 RYVFPLPYL ++++ 273 IYGEKLQFIF+++ 274 KQLDIANYELF ++++ 275 KYGTLDVTF ++++ 276 QYLDVLHAL ++++ 277FYTFPFQQL +++ 279 VWLPASVLF +++ 280 TYNPNLQDKL ++++ 281 NYSPGLVSLIL +++282 NYLVDPVTI +++ 283 EYQEIFQQL +++ 284 DYLKDPVTI +++ 285 VYVGDALLHAI+++ 286 SYGTILSHI ++++ 287 IYNPNLLTASKF +++ 288 VYPDTVALTF ++ 289FFHEGQYVF ++++ 290 KYGDFKLLEF ++++ 291 YYLGSGRETF +++ 292 FYPQIINTF ++++293 VYPHFSTTNLI ++++ 294 RFPVQGTVTF +++ 295 SYLVIHERI +++ 296 SYQVIFQHF++++ 297 TYIDTRTVF ++++ 298 AYKSEVVYF ++++ 299 KYQYVLNEF +++ 300TYPSQLPSL +++ 301 KFDDVTMLF ++++ 302 LYLPVHYGF +++ 303 LYSVIKEDF +++ 304EYNEVANLF +++ 305 NYENKQYLF ++++ 306 VYPAEQPQI +++ 307 GYAFTLPLF +++ 308TFDGHGVFF +++ 309 KYYRQTLLF ++ 310 IYAPTLLVF +++ 311 EYLQNLNHI ++++ 312SYTSVLSRL +++ 313 KYTHFIQSF ++++ 314 RYFKGDYSI +++ 315 FYIPHVPVSF +++316 VYFEGSDFKF +++ 317 VFDTSIAQLF +++ 318 TYSNSAFQYF +++ 319 KYSDVKNLI++++ 341 SYLTQHQRI +++ 342 NYAFLHRTL +++ 343 NYLGGTSTI +++ 344 EYNSDLHQF+++ 345 EYNSDLHQFF +++ 347 VYAEVNSL +++ 348 IYLEHTESI +++ 349 QYSIISNVF+++ 350 KYGNFIDKL +++ 351 IFHEVPLKF +++ 352 QYGGDLTNTF +++ 353 TYGKIDLGF+++ 354 VYNEQIRDLL +++ 355 IYVTGGHLF +++ 356 NYMPGQLTI ++++ 357QFITSTNTF ++++ 358 YYSEVPVKL +++ 359 NYGVLHVTF ++++ 360 VFSPDGHLF +++361 TYADIGGLDNQI +++ 362 VYNYAEQTL ++ 363 SYAELGTTI ++ 364 KYLNENQLSQL+++ 365 VFIDHPVHL ++++ 366 QYLELAHSL +++ 367 LYQDHMQYI ++ 368 KYQNVKHNL+++ 369 VYTHEVVTL +++ 370 RFIGIPNQF +++ 371 AYSHLRYVF ++ 372 VYVIEPHSMEF+++ 373 GYISNGELF +++ 374 VFLPRVTEL ++ 375 KYTDYILKI +++ 376 VYTPVASRQSL+++ 377 QYTPHSHQF +++ 378 VYIAELEKI +++ 379 VFIAQGYTL ++++ 380 VYTGIDHHW++++ 381 KYPASSSVF +++ 382 AYLPPLQQVF +++ 383 RYKPGEPITF +++ 384RYFDVGLHNF +++ 385 QYIEELQKF +++ 386 TFSDVEAHF +++ 387 KYTEKLEEI +++ 388IYGEKTYAF +++

Example 6

Absolute Quantitation of Tumor Associated Peptides Presented on the CellSurface

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor-associated and—specific peptides,selection criteria include but are not restricted to exclusiveness ofpresentation and the density of peptide presented on the cell surface.In addition to the isolation and relative quantitation of peptides asdescribed in Example 1, the inventors did analyze absolute peptidecopies per cell as described. The quantitation of TUMAP copies per cellin solid tumor samples requires the absolute quantitation of theisolated TUMAP, the efficiency of TUMAP isolation, and the cell count ofthe tissue sample analyzed.

Peptide Quantitation by nanoLC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each peptide using the internalstandard method. The internal standard is a double-isotope-labeledvariant of each peptide, i.e. two isotope-labeled amino acids wereincluded in TUMAP synthesis. It differs from the tumor-associatedpeptide only in its mass but shows no difference in otherphysicochemical properties (Anderson et al., 2012). The internalstandard was spiked to each MS sample and all MS signals were normalizedto the MS signal of the internal standard to level out potentialtechnical variances between MS experiments.

The calibration curves were prepared in at least three differentmatrices, i.e. HLA peptide eluates from natural samples similar to theroutine MS samples, and each preparation was measured in duplicate MSruns. For evaluation, MS signals were normalized to the signal of theinternal standard and a calibration curve was calculated by logisticregression.

For the quantitation of tumor-associated peptides from tissue samples,the respective samples were also spiked with the internal standard, theMS signals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide/MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide/MHC complexes, single-isotope-labeled versions of the TUMAPswere used, i.e. one isotope-labeled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide/MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labeled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a low number of samples andwas comparable among these tissue samples. In contrast, the isolationefficiency differs between individual peptides. This suggests that theisolation efficiency, although determined in only a limited number oftissue samples, may be extrapolated to any other tissue preparation.However, it is necessary to analyze each TUMAP individually as theisolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected toabsolute peptide quantitation, the inventors applied DNA contentanalysis. This method is applicable to a wide range of samples ofdifferent origin and, most importantly, frozen samples (Alcoser et al.,2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During thepeptide isolation protocol, a tissue sample is processed to a homogenouslysate, from which a small lysate aliquot is taken. The aliquot isdivided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit,Qiagen, Hilden, Germany). The total DNA content from each DNA isolationis quantified using a fluorescence-based DNA quantitation assay (QubitdsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at leasttwo replicates.

In order to calculate the cell number, a DNA standard curve fromaliquots of single healthy blood cells, with a range of defined cellnumbers, has been generated. The standard curve is used to calculate thetotal cell content from the total DNA content from each DNA isolation.The mean total cell count of the tissue sample used for peptideisolation is extrapolated considering the known volume of the lysatealiquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell number for selected peptides is shown inTable 15.

TABLE 15 Absolute copy numbers. The table lists the results of absolutepeptide quantitation in tumor samples. The median number of copies percell are indicated for each peptide: <100 = +; >=100 = ++; >=1,000+++; >=10,000 = ++++. The number of samples, in which evaluable, highquality MS data are available is indicated. SEQ ID Copies per cellNumber of No. Peptide Code (median) samples 70 DNMT3B-001 ++ 16 323KIAA0226L-002 ++ 19 325 ZNF-003 ++ 14

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1. A peptide consisting of the amino acid sequence LYLENNAQTQF (SEQ IDNO: 193) in the form of a pharmaceutically acceptable salt.
 2. Thepeptide of claim 1, wherein said peptide has the ability to bind to anMHC class-I molecule, and wherein said peptide, when bound to said MHC,is capable of being recognized by CD8 T cells.
 3. The peptide of claim1, wherein the pharmaceutically acceptable is chloride salt.
 4. Thepeptide of claim 1, wherein the pharmaceutically acceptable is acetatesalt.
 5. A composition comprising the peptide of claim 1, wherein thecomposition comprises an adjuvant and a pharmaceutically acceptablecarrier.
 6. The composition of claim 5, wherein the peptide is in theform of a chloride salt.
 7. The composition of claim 5, wherein thepeptide is in the form of an acetate salt.
 8. The composition of claim 5wherein the adjuvant is selected from the group consisting of anti-CD40antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib,bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides andderivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulateformulations with poly(lactide co-glycolide) (PLG), virosomes,interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, andIL-23.
 9. The composition of claim 8, wherein the adjuvant is IL-2. 10.The composition of claim 8, wherein the adjuvant is IL-7.
 11. Thecomposition of claim 8, wherein the adjuvant is IL-12.
 12. Thecomposition of claim 8, wherein the adjuvant is IL-15.
 13. Thecomposition of claim 8, wherein the adjuvant is IL-21.
 14. A pegylatedpeptide consisting of the amino acid sequence of LYLENNAQTQF (SEQ ID NO:193) or a pharmaceutically acceptable salt thereof.
 15. The peptide ofclaim 14, wherein the pharmaceutically acceptable is chloride salt. 16.The peptide of claim 14, wherein the pharmaceutically acceptable isacetate salt.
 17. A composition comprising the pegylated peptide ofclaim 14 or pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 18. A peptide consisting of theamino acid sequence of LYLENNAQTQF (SEQ ID NO: 193), wherein at leastone amino acid of the peptide is a D-amino acid.
 19. The peptide in theform of a pharmaceutically acceptable salt of claim 1, wherein saidpeptide is produced by solid phase peptide synthesis or produced by ayeast cell or bacterial cell expression system.
 20. A compositioncomprising the peptide of claim 1, wherein the composition is apharmaceutical composition and comprises water and a buffer.